Nucleic acid molecules targeting superoxide dismutase 1 (sod1)

ABSTRACT

Aspects of the invention relate to methods for treating ALS comprising administering to a subject in need thereof a therapeutically effective amount of a nucleic acid molecule that is directed against a gene encoding superoxide dismutase 1 (SOD1).

RELATED APPLICATIONS

This application claims the benefit under 35 U.S.C. § 119(e) of U.S.Provisional Application Ser. No. 62/189,050, filed on Jul. 6, 2015,entitled “NUCLEIC ACID MOLECULES TARGETING SUPEROXIDE DISMUTASE 1(SOD1)”, the entire contents of which are incorporated herein byreference.

FIELD OF THE INVENTION

The disclosure relates, at least in part, to the use of nucleic acidmolecules with improved in vivo delivery properties targeting SOD1 forthe treatment of neurological disorders such as amyotrophic lateralsclerosis (ALS).

BACKGROUND

Complementary oligonucleotide sequences are promising therapeutic agentsand useful research tools in elucidating gene functions. However, priorart oligonucleotide molecules suffer from several problems that mayimpede their clinical development, and frequently make it difficult toachieve intended efficient inhibition of gene expression (includingprotein synthesis) using such compositions in vivo.

A major problem has been the delivery of these compounds to cells andtissues. Conventional double-stranded RNAi compounds, 19-29 bases long,form a highly negatively-charged rigid helix of approximately 1.5 by10-15 nm in size. This rod type molecule cannot get through thecell-membrane and as a result has very limited efficacy both in vitroand in vivo. As a result, all conventional RNAi compounds require somekind of delivery vehicle to promote their tissue distribution andcellular uptake. This is considered to be a major limitation of the RNAitechnology.

There have been previous attempts to apply chemical modifications tooligonucleotides to improve their cellular uptake properties. One suchmodification was the attachment of a cholesterol molecule to theoligonucleotide. A first report on this approach was by Letsinger etal., in 1989. Subsequently, ISIS Pharmaceuticals, Inc. (Carlsbad,Calif.) reported on more advanced techniques in attaching thecholesterol molecule to the oligonucleotide (Manoharan, 1992).

With the discovery of siRNAs in the late nineties, similar types ofmodifications were attempted on these molecules to enhance theirdelivery profiles. Cholesterol molecules conjugated to slightly modified(Soutschek, 2004) and heavily modified (Wolfrum, 2007) siRNAs appearedin the literature. Yamada et al., 2008 also reported on the use ofadvanced linker chemistries which further improved cholesterol mediateduptake of siRNAs. In spite of all this effort, the uptake of these typesof compounds impaired to be inhibited in the presence of biologicalfluids resulting in highly limited efficacy in gene silencing in vivo,limiting the applicability of these compounds in a clinical setting.

SUMMARY

In some aspects, the disclosure relates to an isolated double strandednucleic acid molecule directed agains superoxide dismutase 1 (SOD1)comprising a guide strand and a passenger strand, wherein the isolateddouble stranded nucleic acid molecule includes a double stranded regionand a single stranded region, wherein the region of the molecule that isdouble stranded is from 8-15 nucleotides long, wherein the guide strandcontains a single stranded region that is 2-14 nucleotides long, whereinthe guide strand contains 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, 20, 21 or 22 phosphorothioate modifications, whereinthe passenger strand is 8 to 15 nucleotides long, wherein the passengerstrand contains 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14phosphorothioate modifications, and wherein at least 40% of thenucleotides of the isolated double stranded nucleic acid molecule aremodified.

In some embodiments, at least 60% of the nucleotides are modified. Insome embodiments, at least one strand of the isolated double strandednucleic acid molecule comprises a completely phosphorothioated backbone.In some embodiments, at least one strand of the isolated double strandednucleic acid molecule is completely phosphorothioated, or is completelyphosphorothioated with the exception of one residue. In someembodiments, at least one of the nucleotides of the isolated doublestranded nucleic acid molecule that is modified comprises a 2′O-methylor a 2′-fluoro modification.

In some embodiments, the isolated double stranded nucleic acid moleculeis directed against superoxide dismutase 1 (SOD1). In some embodiments,the isolated double stranded nucleic acid molecule does not comprise themodification pattern of SEQ ID NO: 40 described in PCT Publicationnumber WO2010/033247.

In some embodiments, a plurality of the U's and/or C's include ahydrophobic modification, selected from the group consisting of methyl,isobutyl, octyl, imidazole or thiophene, wherein the modifications arelocated on positions 4 or 5 of U's and/or C's.

In some embodiments, the isolated double stranded nucleic acid moleculecomprises at least 12 contiguous nucleotides of a sequence selected fromthe sequences within Tables 1-8, including the modification patternprovided in Tables 1-8.

In some aspects, the disclosure relates to an isolated double strandednucleic acid molecule that comprises at least 12 contiguous nucleotidesof a sequence selected from the sequences within Tables 1-8, wherein ifthe isolated double stranded nucleic acid molecule comprises at least 12contiguous nucleotides of a sequence selected from SEQ ID NOs: 70, 71,72, 73, 79, 80, 81, or 84 in Table 2, then the guide strand containsmore than 6 phosphorothioate modifications. In some embodiments, theisolated double stranded nucleic acid molecule further comprises ahydrophobic conjugate that is attached to the isolated double strandednucleic acid molecule.

In some embodiments, the sense strand of the isolated nucleic acidmolecule comprises at least 12 consecutive nucleotides of SEQ ID NO: 2,SEQ ID NO: 32, or SEQ ID NO: 122, and the guide strand comprises atleast 12 consecutive nucleotides of SEQ ID NO: 61, SEQ ID NO: 91, or SEQID NO: 123. In some embodiments, the sense strand of the isolatednucleic acid molecule comprises SEQ ID NO: 2, SEQ ID NO: 32, or SEQ IDNO: 122, and the guide strand comprises SEQ ID NO: 61, SEQ ID NO: 91, orSEQ ID NO: 123.

In some embodiments, the sense strand of the isolated nucleic acidmolecule comprises at least 12 consecutive nucleotides of SEQ ID NO: 4,SEQ ID NO: 34, or SEQ ID NO: 126, and the guide strand comprises atleast 12 consecutive nucleotides of SEQ ID NO: 63 or SEQ ID NO: 93. Insome embodiments, the sense strand of the isolated nucleic acid moleculecomprises SEQ ID NO: 4, SEQ ID NO: 34, or SEQ ID NO: 126, and the guidestrand comprises SEQ ID NO: 63 or SEQ ID NO: 93.

In some embodiments, the sense strand of the isolated nucleic acidmolecule comprises at least 12 consecutive nucleotides of SEQ ID NO: 9,SEQ ID NO: 38, or SEQ ID NO: 135, and the guide strand comprises atleast 12 consecutive nucleotides of SEQ ID NO: 68, SEQ ID NO: 97, or SEQID NO: 136. In some embodiments, the sense strand of the isolatednucleic acid molecule comprises SEQ ID NO: 9, SEQ ID NO: 38, or SEQ IDNO:135, and the guide strand comprises SEQ ID NO: 68, SEQ ID NO: 97, orSEQ ID NO: 136.

In some embodiments, the sense strand of the isolated nucleic acidmolecule comprises at least 12 consecutive nucleotides of SEQ ID NO: 10or SEQ ID NO: 39, and the guide strand comprises at least 12 consecutivenucleotides of SEQ ID NO: 69 or SEQ ID NO: 98. In some embodiments, thesense strand of the isolated nucleic acid molecule comprises SEQ ID NO:10 or SEQ ID NO: 39, and the guide strand comprises SEQ ID NO: 69 or SEQID NO: 98.

In some embodiments, the sense strand of the isolated double strandednucleic acid molecule comprises at least 12 consecutive nucleotides ofSEQ ID NO: 5, SEQ ID NO: 127 or SEQ ID NO: 137, and the guide strandcomprises at least 12 consecutive nucleotides of SEQ ID NO: 64, SEQ IDNO: 128 or SEQ ID NO: 138. In some embodiments, the sense strand of theisolated nucleic acid molecule comprises SEQ ID NO: 5, SEQ ID NO: 127 orSEQ ID NO: 137, and the guide strand comprises SEQ ID NO: 64, SEQ ID NO:128 or SEQ ID NO: 138.

In some embodiments, the isolated double stranded nucleic acid does notform a hairpin.

In some aspects, the disclosure relates to a composition comprising anisolated double stranded nucleic acid molecule as described by thedisclosure. In some embodiments, the composition comprises an excipient(e.g., a pharmaceutically acceptable carrier). In some embodiments, thecomposition comprises a second therapeutic agent, such as a nucleic acid(e.g., sd-rxRNA, etc.), small molecule, peptide, or polypeptide (e.g.,antibody).

In some aspects, the disclosure relates to a method for treating ALScomprising administering to a subject in need thereof a therapeuticallyeffective amount of a nucleic acid molecule that is directed against agene encoding superoxide dismutase 1 (SOD1).

In some aspects, the disclosure relates to a method for treating ALScomprising administering to a subject in need thereof a therapeuticallyeffective amount of an isolated double stranded nucleic acid moleculedirected against superoxide dismutase 1 (SOD1) comprising a guide strandand a passenger strand, wherein the isolated double stranded nucleicacid molecule includes a double stranded region and a single strandedregion, wherein the region of the molecule that is double stranded isfrom 8-15 nucleotides long, wherein the guide strand contains a singlestranded region that is 2-14 nucleotides long, wherein the guide strandcontains 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,20, 21 or 22 phosphorothioate modifications, wherein the passengerstrand is 8 to 15 nucleotides long, wherein the passenger strandcontains 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14 phosphorothioatemodifications, wherein at least 40% of the nucleotides of the isolateddouble stranded nucleic acid molecule are modified, and wherein theisolated double stranded nucleic acid molecule comprises at least 12contiguous nucleotides of a sequence selected from the sequences withinTables 1-8, including the modification pattern provided in Tables 1-8.

In some embodiments, the isolated double stranded nucleic acid moleculeis completely phosphorothioated, or is completely phosphorothioated withthe exception of one residue In some embodiments, at least one of thenucleotides of the isolated double stranded nucleic acid molecule thatis modified comprises a 2′O-methyl or a 2′-fluoro modification.

In some embodiments, the isolated double stranded nucleic acid moleculefurther comprises a hydrophobic conjugate that is attached to theisolated double stranded nucleic acid molecule.

In some embodiments, the isolated double stranded nucleic acid moleculeis formulated for delivery to the central nervous system.

In some embodiments, the isolated double stranded nucleic acid moleculeis administered via intrathecal infusion and/or injection.

In some embodiments of methods described herein, the sense strand of theisolated nucleic acid molecule comprises at least 12 consecutivenucleotides of SEQ ID NO: 2, SEQ ID NO: 32, or SEQ ID NO: 122, and theguide strand comprises at least 12 consecutive nucleotides of SEQ ID NO:61, SEQ ID NO: 91, or SEQ ID NO: 123.

In some embodiments of methods described herein, the sense strand of theisolated nucleic acid molecule comprises SEQ ID NO: 2, SEQ ID NO: 32, orSEQ ID NO: 122, and the guide strand comprises SEQ ID NO: 61, SEQ ID NO:91, or SEQ ID NO: 123.

In some embodiments of methods described herein, the sense strand of theisolated nucleic acid molecule comprises at least 12 consecutivenucleotides of SEQ ID NO: 4, SEQ ID NO: 34, or SEQ ID NO: 126, and theguide strand comprises at least 12 consecutive nucleotides of SEQ ID NO:63 or SEQ ID NO: 93.

In some embodiments of methods described herein, the sense strand of theisolated nucleic acid molecule comprises SEQ ID NO: 4, SEQ ID NO: 34, orSEQ ID NO: 126, and the guide strand comprises SEQ ID NO: 63 or SEQ IDNO: 93.

In some embodiments of methods described herein, the sense strand of theisolated nucleic acid molecule comprises at least 12 consecutivenucleotides of SEQ ID NO: 9, SEQ ID NO: 38, or SEQ ID NO:135, and theguide strand comprises at least 12 consecutive nucleotides of SEQ ID NO:68, SEQ ID NO: 97, or SEQ ID NO: 136.

In some embodiments of methods described herein, the sense strand of theisolated nucleic acid molecule comprises SEQ ID NO: 9, SEQ ID NO: 38, orSEQ ID NO: 135, and the guide strand comprises SEQ ID NO: 68, SEQ ID NO:97, or SEQ ID NO: 136.

In some embodiments of methods described herein, the sense strand of theisolated nucleic acid molecule comprises at least 12 consecutivenucleotides of SEQ ID NO: 10 or SEQ ID NO: 39, and the guide strandcomprises at least 12 consecutive nucleotides of SEQ ID NO: 69 or SEQ IDNO: 98.

In some embodiments of methods described herein, the sense strand of theisolated nucleic acid molecule comprises SEQ ID NO: 10 or SEQ ID NO: 39,and the guide strand comprises SEQ ID NO: 69 or SEQ ID NO: 98.

In some embodiments of methods described herein, the sense strand of theisolated double stranded nucleic acid molecule comprises at least 12consecutive nucleotides of SEQ ID NO: 5, SEQ ID NO: 127 or SEQ ID NO:137, and the guide strand comprises at least 12 consecutive nucleotidesof SEQ ID NO: 64, SEQ ID NO: 128 or SEQ ID NO: 138.

In some embodiments of methods described herein, the sense strand of theisolated nucleic acid molecule comprises SEQ ID NO: 5, SEQ ID NO: 127 orSEQ ID NO: 137, and the guide strand comprises SEQ ID NO: 64, SEQ ID NO:128 or SEQ ID NO: 138.

Each of the limitations of the invention can encompass variousembodiments of the invention. It is, therefore, anticipated that each ofthe limitations of the invention involving any one element orcombinations of elements can be included in each aspect of theinvention. This invention is not limited in its application to thedetails of construction and the arrangement of components set forth inthe following description or illustrated in the drawings. The inventionis capable of other embodiments and of being practiced or of beingcarried out in various ways.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Reducing phosphorothioate content results in active ps-rxRNAvariants with reduced cellular toxicity in vitro. The upper panel showsSOD1 silencing and the lower panel shows cell viability. These datademonstrate that ps-rxRNA variants 25635 and 25637 are potent and have amore favorable cellular toxicity profile (in vitro) compared to 25600.

FIG. 2. Reducing phosphorothioate content results in active ps-rxRNAvariants with reduced cellular toxicity in vitro. The upper panel showsSOD1 silencing and the lower panel shows cell viability. These data showthat ps-rxRNA variant 25645 is potent and has a slightly more favorablecellular toxicity profile (in vitro) compared to 25600.

FIG. 3. Brain-cerebellum (IC injection in rat). Penetration increaseswith change in chemistry content; uptake 25635<25637<25645<25652. *Frontal cortex, not cerebellum.

FIG. 4. Cervical spinal cord-transverse cut. 25637 and var 25645 aresimilar but both are less than 25652 in distribution.

FIG. 5. Cervical spinal cord-longitudinal cut. 25637 is less than 25652in distribution into the tissue. Administration of original fl-ps-rxRNAcompound results in full brain and spinal cord penetration. ps-rxRNAvariant 1 (25635) in this particular assay was insufficient to achievebrain or spinal cord penetration. ps-rxRNA variants 2 and 3 (25637 and25645, respectively) both result in uptake by cells of the brain andspinal cord but less than original fl-ps-rxRNA (25652).

FIG. 6. Reduction of SOD1 mRNA following a 14-day intrathecal infusionof SOD targeting sd-rxRNA in C57BL/6 normal (non-transgenic) mice usingan implanted osmotic pump. 14 day silencing with Oligo ID 25652 innormal mice. 14 day osmotic pumps were filled with 100 μl of 10 mg/ml ofeach compound. Gene expression analysis by qPCR was normalized to thePPIB housekeeping gene and was plotted relative to SOD1 expression inthe PBS group. Silencing was observed in the lumbar region of the spinalcord only (region of catheter placement). The pump flow rate was 0.25μl/hr, therefore 60 μg of SOD1 targeting or non-targeting control (NTC)ps-rxRNA was delivered each day for 14 days (840 μg total). 24 C57BL/6Jmice were used for this study including 8 for Oligo ID 25652: 8 for NTCps-rxRNA; and 8 for PBS.

FIG. 7. Reduction of SOD1 mRNA following a 14-day intrathecal infusionof SOD1 targeting sd-rxRNA Variant 3 (Oligo ID 25645) in normal mice. 14day intrathecal infusion was performed with implanted osmotic pump inC57BL/6 normal (non-transgenic) mice. 14 day osmotic pumps were filledwith 100 μl of 10 mg/ml of each compound. 60 μg of SOD1 targeting ornon-targeting control (NTC) sd-rxRNA variant were administered per day.Gene expression analysis by qPCR was performed to normalize geneexpression to the PPIB housekeeping gene and plotted relative to SOD1expression in the PBS group. Silencing was observed in the lumbar regionof the spinal cord only (region of catheter placement). The pump flowrate was 0.25 l/hr; therefore 60 μg was delivered each day for 14 days(840 μg total). 30 C57BL/6J mice were used for this study, including 10for Oligo ID 25645; 10 for NTC ps-rxRNA; and 10 for PBS.

FIG. 8 depicts data generated using SOD1 sd-rxRNA variant octylmodifications.

FIG. 9 depicts data generated using SOD1 sd-rxRNA variant octylmodifications.

FIG. 10 depicts data generated using SOD1 sd-rxRNA variant thiophenemodifications.

FIG. 11 depicts data generated using SOD1 sd-rxRNA variant isobutylmodifications.

DETAILED DESCRIPTION SOD1 (Copper/Zinc Superoxide Dismutase)

As used herein, “SOD1” refers to the Superoxide Dismutase 1 enzyme,which is one of three superoxide dismutases involved in convertingharmful superoxide radicals to water. Approximately 10% of all ALS casesare dominantly inherited, and of these ˜20% are due to defects incytosolic superoxide dismutase 1 (SOD1). In addition, SOD1 has beenimplicated in non-familial (e.g. sporadic) forms of ALS. (Jones, C. T.,Brock, D. J. H., Chancellor, A. M., Warlow, C. P., Swingler, R. J. Cu/Znsuperoxide dismutase (SOD) mutations and sporadic amyotrophic lateralsclerosis. Lancet 342: 1050-1051, 1993). Without wishing to be bound byany theory, several lines of investigation have suggested that themutations in SOD1 do not cause ALS through loss of the dismutaseactivity of this enzyme. Rather, mutant SOD1 is neurotoxic throughmultiple alternate mechanisms, many of which entail conformationalinstability and aberrant binding and aggregation of the mutant protein.While the precise details of the toxicity of mutant SOD1 are not fullydefined, it is abundantly clear that reduction of the burden of mutantSOD1 protein in animal models significantly delays death. This has beenachieved using both antisense oligonucleotides (ASO) (Smith et al) andsiRNA (Maxwell, Pasinelli et al. 2004) (Xia, Zhou et al. 2006) (Wang,Ghosh et al. 2008). These studies illustrate the principle thatsiRNA-based drugs represent a potentially significant therapeuticadvance for the treatment of ALS and many other CNS disorders. In bothcases, efficacy was achieved by delivery of high amounts of materialover long periods of time; these studies illustrate the point that amajor limitation of ASO and siRNA therapies in their present forms isthe lack of optimally efficient and non-toxic in vivo delivery systems(Smith, Miller et al. 2006; Wang, Ghosh et al. 2008).

Amyotrophic Lateral Sclerosis (ALS)

ALS is a progressive neurodegenerative disease affecting motor neuronsin the central nervous system. Degeneration of the motor neurons resultsin paralysis and eventual death, usually due to respiratory failure. Ina subset of cases, ALS is caused by dominantly transmitted mutations inthe gene encoding cytosolic superoxide dismutase (SOD1). Transgenicexpression of mutant SOD1 causes ALS in mice.

Nucleic Acid Molecules

As used herein, “nucleic acid molecule” includes but is not limited to:sd-rxRNA, sd-rxRNA variant, rxRNAori, oligonucleotides, ASO, siRNA,shRNA, miRNA, ncRNA, cp-lasiRNA, aiRNA, single-stranded nucleic acidmolecules, double-stranded nucleic acid molecules, RNA and DNA. In someembodiments, the nucleic acid molecule is a chemically modified nucleicacid molecule, such as a chemically modified oligonucleotide.

sd-rxRNA Molecules

Aspects of the invention relate to sd-rxRNA molecules. As used herein,an “sd-rxRNA” or an “sd-rxRNA molecule” refers to a self-delivering RNAmolecule such as those described in, and incorporated by reference from,U.S. Pat. No. 8,796,443, granted on Aug. 5, 2014, entitled “REDUCED SIZESELF-DELIVERING RNAI COMPOUNDS”, U.S. Pat. No. 9,175,289, granted onNov. 3, 2015, entitled “REDUCED SIZE SELF-DELIVERING RNAI COMPOUNDS”,and PCT Publication No. WO2010/033247 (Application No.PCT/US2009/005247), filed on Sep. 22, 2009, and entitled “REDUCED SIZESELF-DELIVERING RNAI COMPOUNDS.” Briefly, an sd-rxRNA, (also referred toas an sd-rxRNA^(nano)) is an isolated asymmetric double stranded nucleicacid molecule comprising a guide strand, with a minimal length of 16nucleotides, and a passenger strand of 8-18 nucleotides in length,wherein the double stranded nucleic acid molecule has a double strandedregion and a single stranded region, the single stranded region having4-12 nucleotides in length and having at least three nucleotide backbonemodifications. In preferred embodiments, the double stranded nucleicacid molecule has one end that is blunt or includes a one or twonucleotide overhang. sd-rxRNA molecules can be optimized throughchemical modification, and in some instances through attachment ofhydrophobic conjugates. In some embodiments, the isolated doublestranded nucleic acid molecule does not comprise the modificationpattern of SEQ ID NO: 40 described in PCT Publication numberWO2010/033247.

In some embodiments, an sd-rxRNA comprises an isolated double strandednucleic acid molecule comprising a guide strand and a passenger strand,wherein the region of the molecule that is double stranded is from 8-15nucleotides long, wherein the guide strand contains a single strandedregion that is 4-12 nucleotides long, wherein the single stranded regionof the guide strand contains 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12phosphorothioate modifications, and wherein at least 40% of thenucleotides of the double stranded nucleic acid are modified.

In some embodiments, an sd-rxRNA variant comprises an sd-rxRNA thatcontains an 18 to 23 nucleotide long guide strand (antisense strand) anda 10 to 15 nucleotide long passenger strand (sense strand). The guidestrand and passenger strand can form an asymmetric duplex. In someembodiments, the guide strand contains between 6 to 22 backbonemodifications, including phosphorothioate modifications. In someembodiments, the passenger strand contains between 2 to 14 backbonemodifications, including phosphorothioate modifications. In someembodiments, the passenger strand is attached to a hydrophobicconjugate. The term “sd-rxRNA variant” is used interchangeably hereinwith “ps-rxRNA”.

In some embodiments, the guide strand contains 2, 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 or 22 phosphorothioatemodifications. In some embodiments, the guide strand is completelyphosphorothioated. In some embodiments, the passenger strand contains 3,4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14 phosphorothioate modifications.In some embodiments, the passenger strand is completelyphosphorothioated. It was surprisingly found herein that high levels ofphosphorothioate modifications can lead to increased delivery ofisolated double stranded nucleic acid molecules.

Nucleic acid molecules associated with the invention are also referredto herein as polynucleotides, isolated double stranded or duplex nucleicacids, oligonucleotides, nano molecules, nano RNA, sd-rxRNA^(nano),sd-rxRNA or RNA molecules of the invention.

sd-rxRNAs are much more effectively taken up by cells compared toconventional siRNAs. These molecules are highly efficient in silencingof target gene expression and offer significant advantages overpreviously described RNAi molecules including high activity in thepresence of serum, efficient self-delivery, compatibility with a widevariety of linkers, and reduced presence or complete absence of chemicalmodifications that are associated with toxicity.

In contrast to single-stranded polynucleotides, duplex polynucleotideshave traditionally been difficult to deliver to a cell as they haverigid structures and a large number of negative charges which makesmembrane transfer difficult. sd-rxRNAs however, although partiallydouble-stranded, are recognized in vivo as single-stranded and, as such,are capable of efficiently being delivered across cell membranes. As aresult the polynucleotides of the invention are capable in manyinstances of self-delivery. Thus, the polynucleotides of the inventionmay be formulated in a manner similar to conventional RNAi agents orthey may be delivered to the cell or subject alone (or with non-deliverytype carriers) and allowed to self-deliver. In one embodiment of thepresent invention, self-delivering asymmetric double-stranded RNAmolecules are provided in which one portion of the molecule resembles aconventional RNA duplex and a second portion of the molecule is singlestranded.

The oligonucleotides of the invention in some aspects have a combinationof asymmetric structures including a double stranded region and a singlestranded region of 5 nucleotides or longer, specific chemicalmodification patterns and are conjugated to lipophilic or hydrophobicmolecules. This class of RNAi like compounds have superior efficacy invitro and in vivo. It is believed that the reduction in the size of therigid duplex region in combination with phosphorothioate modificationsapplied to a single stranded region contribute to the observed superiorefficacy.

In some embodiments an RNAi compound associated with the inventioncomprises an asymmetric compound comprising a duplex region (requiredfor efficient RISC entry of 8-15 bases long) and single stranded regionof 4-12 nucleotides long. In some embodiments, the duplex region is 13or 14 nucleotides long. A 6 or 7 nucleotide single stranded region ispreferred in some embodiments. In some embodiments, the RNAi compoundcomprises 2-12 phosphorothioate internucleotide linkages (referred to asphosphorothioate modifications). 6-8 phosphorothioate internucleotidelinkages are preferred in some embodiments. In some embodiments, theRNAi compounds include a unique chemical modification pattern, whichprovides stability and is compatible with RISC entry. The combination ofthese elements has resulted in unexpected properties which are highlyuseful for delivery of RNAi reagents in vitro and in vivo.

The chemical modification pattern, which provides stability and iscompatible with RISC entry includes modifications to the sense, orpassenger, strand as well as the antisense, or guide, strand. Forinstance the passenger strand can be modified with any chemical entitieswhich confirm stability and do not interfere with activity. Suchmodifications include 2′ ribo modifications (O-methyl, 2′ F, 2 deoxy andothers) and backbone modification like phosphorothioate modifications. Apreferred chemical modification pattern in the passenger strand includesO-methyl modification of C and U nucleotides within the passenger strandor alternatively the passenger strand may be completely O-methylmodified.

The guide strand, for example, may also be modified by any chemicalmodification which confirms stability without interfering with RISCentry. A preferred chemical modification pattern in the guide strandincludes the majority of C and U nucleotides being 2′ F modified and the5′ end being phosphorylated. Another preferred chemical modificationpattern in the guide strand includes 2′O-methyl modification of position1 and C/U in positions 11-18 and 5′ end chemical phosphorylation. Yetanother preferred chemical modification pattern in the guide strandincludes 2′O-methyl modification of position 1 and C/U in positions11-18 and 5′ end chemical phosphorylation and 2′F modification of C/U inpositions 2-10. In some embodiments the passenger strand and/or theguide strand contains at least one 5-methyl C or U modifications.

In some embodiments, at least 30% of the nucleotides in the sd-rxRNA aremodified. For example, at least 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%,38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%/o, 50%, 51%,52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%,66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%,80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98% or 99% of the nucleotides in the sd-rxRNA aremodified. In some embodiments, 100% of the nucleotides in the sd-rxRNAare modified.

The above-described chemical modification patterns of theoligonucleotides of the invention are well tolerated and actuallyimproved efficacy of asymmetric RNAi compounds. In some embodiments,elimination of any of the described components (Guide strandstabilization, phosphorothioate stretch, sense strand stabilization andhydrophobic conjugate) or increase in size in some instances results insub-optimal efficacy and in some instances complete loss of efficacy.The combination of elements results in development of a compound, whichis fully active following passive delivery to cells such as HeLa cells.

The sd-rxRNA can be further improved in some instances by improving thehydrophobicity of compounds using of novel types of chemistries. Forexample, one chemistry is related to use of hydrophobic basemodifications. Any base in any position might be modified, as long asmodification results in an increase of the partition coefficient of thebase. The preferred locations for modification chemistries are positions4 and 5 of the pyrimidines. The major advantage of these positions is(a) ease of synthesis and (b) lack of interference with base-pairing andA form helix formation, which are essential for RISC complex loading andtarget recognition. A version of sd-rxRNA compounds where multiple deoxyUridines are present without interfering with overall compound efficacywas used. In addition major improvement in tissue distribution andcellular uptake might be obtained by optimizing the structure of thehydrophobic conjugate. In some of the preferred embodiment the structureof sterol is modified to alter (increase/decrease) C17 attached chain.This type of modification results in significant increase in cellularuptake and improvement of tissue uptake prosperities in vivo.

dsRNA formulated according to the invention also includes rxRNAori.rxRNAori refers to a class of RNA molecules described in andincorporated by reference from PCT Publication No. WO2009/102427(Application No. PCT/US2009/000852), filed on Feb. 11, 2009, andentitled, “MODIFIED RNAI POLYNUCLEOTIDES AND USES THEREOF” and US PatentPublication No. US 2011-0039914 entitled “MODIFIED RNAI POLYNUCLEOTIDESAND USES THEREOF.”

In some embodiments, an rxRNAori molecule comprises a double-strandedRNA (dsRNA) construct of 12-35 nucleotides in length, for inhibitingexpression of a target gene, comprising: a sense strand having a 5′-endand a 3′-end, wherein the sense strand is highly modified with2′-modified ribose sugars, and wherein 3-6 nucleotides in the centralportion of the sense strand are not modified with 2′-modified ribosesugars and, an antisense strand having a 5′-end and a 3′-end, whichhybridizes to the sense strand and to mRNA of the target gene, whereinthe dsRNA inhibits expression of the target gene in a sequence-dependentmanner.

rxRNAori can contain any of the modifications described herein. In someembodiments, at least 30% of the nucleotides in the rxRNAori aremodified. For example, at least 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%,38%, 39, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%,52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%,66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%,80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98% or 99% of the nucleotides in the rxRNAori aremodified. In some embodiments, 100% of the nucleotides in the sd-rxRNAare modified. In some embodiments, only the passenger strand of therxRNAori contains modifications.

This invention is not limited in its application to the details ofconstruction and the arrangement of components set forth in thefollowing description or illustrated in the drawings. The invention iscapable of other embodiments and of being practiced or of being carriedout in various ways. Also, the phraseology and terminology used hereinis for the purpose of description and should not be regarded aslimiting. The use of “including,” “comprising,” or “having,”“containing,” “involving,” and variations thereof herein, is meant toencompass the items listed thereafter and equivalents thereof as well asadditional items.

Aspects of the invention relate to isolated double stranded nucleic acidmolecules comprising a guide (antisense) strand and a passenger (sense)strand. As used herein, the term “double-stranded” refers to one or morenucleic acid molecules in which at least a portion of the nucleomonomersare complementary and hydrogen bond to form a double-stranded region. Insome embodiments, the length of the guide strand ranges from 16-29nucleotides long. In certain embodiments, the guide strand is 16, 17,18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, or 29 nucleotides long. Theguide strand has complementarity to a target gene. Complementaritybetween the guide strand and the target gene may exist over any portionof the guide strand. Complementarity as used herein may be perfectcomplementarity or less than perfect complementarity as long as theguide strand is sufficiently complementary to the target that itmediates RNAi. In some embodiments complementarity refers to less than25%, 20%, 15%, 10%, 5%, 4%, 3%, 2%, or 1% mismatch between the guidestrand and the target. Perfect complementarity refers to 100%complementarity. In some embodiments, siRNA sequences with insertions,deletions, and single point mutations relative to the target sequencehave also been found to be effective for inhibition. Moreover, not allpositions of a siRNA contribute equally to target recognition.Mismatches in the center of the siRNA are most critical and essentiallyabolish target RNA cleavage. Mismatches upstream of the center orupstream of the cleavage site referencing the antisense strand aretolerated but significantly reduce target RNA cleavage. Mismatchesdownstream of the center or cleavage site referencing the antisensestrand, preferably located near the 3′ end of the antisense strand, e.g.1, 2, 3, 4, 5 or 6 nucleotides from the 3′ end of the antisense strand,are tolerated and reduce target RNA cleavage only slightly.

While not wishing to be bound by any particular theory, in someembodiments, the guide strand is at least 16 nucleotides in length andanchors the Argonaute protein in RISC. In some embodiments, when theguide strand loads into RISC it has a defined seed region and targetmRNA cleavage takes place across from position 10-11 of the guidestrand. In some embodiments, the 5′ end of the guide strand is or isable to be phosphorylated. The nucleic acid molecules described hereinmay be referred to as minimum trigger RNA.

In some embodiments, the length of the passenger strand ranges from 8-15nucleotides long. In certain embodiments, the passenger strand is 8, 9,10, 11, 12, 13, 14 or 15 nucleotides long. The passenger strand hascomplementarity to the guide strand. Complementarity between thepassenger strand and the guide strand can exist over any portion of thepassenger or guide strand. In some embodiments, there is 100%complementarity between the guide and passenger strands within thedouble stranded region of the molecule.

Aspects of the invention relate to double stranded nucleic acidmolecules with minimal double stranded regions. In some embodiments theregion of the molecule that is double stranded ranges from 8-15nucleotides long. In certain embodiments, the region of the moleculethat is double stranded is 8, 9, 10, 11, 12, 13, 14 or 15 nucleotideslong. In certain embodiments the double stranded region is 13 or 14nucleotides long. There can be 100% complementarity between the guideand passenger strands, or there may be one or more mismatches betweenthe guide and passenger strands. In some embodiments, on one end of thedouble stranded molecule, the molecule is either blunt-ended or has aone-nucleotide overhang. The single stranded region of the molecule isin some embodiments between 4-12 nucleotides long. For example thesingle stranded region can be 4, 5, 6, 7, 8, 9, 10, 11 or 12 nucleotideslong. However, in certain embodiments, the single stranded region canalso be less than 4 or greater than 12 nucleotides long. In certainembodiments, the single stranded region is at least 6 or at least 7nucleotides long.

RNAi constructs associated with the invention can have a thermodynamicstability (ΔG) of less than −13 kkal/mol. In some embodiments, thethermodynamic stability (ΔG) is less than −20 kkal/mol. In someembodiments there is a loss of efficacy when (ΔG) goes below −21kkal/mol. In some embodiments a (ΔG) value higher than −13 kkal/mol iscompatible with aspects of the invention. Without wishing to be bound byany theory, in some embodiments a molecule with a relatively higher (ΔG)value may become active at a relatively higher concentration, while amolecule with a relatively lower (ΔG) value may become active at arelatively lower concentration. In some embodiments, the (ΔG) value maybe higher than −9 kkcal/mol. The gene silencing effects mediated by theRNAi constructs associated with the invention, containing minimal doublestranded regions, are unexpected because molecules of almost identicaldesign but lower thermodynamic stability have been demonstrated to beinactive (Rana et al 2004).

Without wishing to be bound by any theory, a stretch of 8-10 bp of dsRNAor dsDNA may be structurally recognized by protein components of RISC orco-factors of RISC. Additionally, there is a free energy requirement forthe triggering compound that it may be either sensed by the proteincomponents and/or stable enough to interact with such components so thatit may be loaded into the Argonaute protein. If optimal thermodynamicsare present and there is a double stranded portion that is preferably atleast 8 nucleotides then the duplex will be recognized and loaded intothe RNAi machinery.

In some embodiments, thermodynamic stability is increased through theuse of LNA bases. In some embodiments, additional chemical modificationsare introduced. Several non-limiting examples of chemical modificationsinclude: 5′ Phosphate, 2′-O-methyl, 2′-O-ethyl, 2′-fluoro,ribothymidine, C-5 propynyl-dC (pdC) and C-5 propynyl-dU (pdU); C-5propynyl-C(pC) and C-5 propynyl-U (pU); 5-methyl C, 5-methyl U, 5-methyldC, 5-methyl dU methoxy, (2,6-diaminopurine),5′-Dimethoxytrityl-N4-ethyl-2′-deoxyCytidine and MGB (minor groovebinder). It should be appreciated that more than one chemicalmodification can be combined within the same molecule.

Molecules associated with the invention are optimized for increasedpotency and/or reduced toxicity. For example, nucleotide length of theguide and/or passenger strand, and/or the number of phosphorothioatemodifications in the guide and/or passenger strand, can in some aspectsinfluence potency of the RNA molecule, while replacing 2′-fluoro (2′F)modifications with 2′-O-methyl (2′OMe) modifications can in some aspectsinfluence toxicity of the molecule. Specifically, reduction in 2′Fcontent of a molecule is predicted to reduce toxicity of the molecule.Furthermore, the number of phosphorothioate modifications in an RNAmolecule can influence the uptake of the molecule into a cell, forexample the efficiency of passive uptake of the molecule into a cell.Preferred embodiments of molecules described herein have no 2′Fmodification and yet are characterized by equal efficacy in cellularuptake and tissue penetration. Such molecules represent a significantimprovement over prior art, such as molecules described by Accell andWolfrum, which are heavily modified with extensive use of 2′F.

In some embodiments, a guide strand is approximately 18-19 nucleotidesin length and has approximately 2-14 phosphate modifications. Forexample, a guide strand can contain 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14 or more than 14 nucleotides that are phosphate-modified. Theguide strand may contain one or more modifications that confer increasedstability without interfering with RISC entry. The phosphate modifiednucleotides, such as phosphorothioate modified nucleotides, can be atthe 3′ end, 5′ end or spread throughout the guide strand. In someembodiments, the 3′ terminal 10 nucleotides of the guide strand contains1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 phosphorothioate modified nucleotides.The guide strand can also contain 2′F and/or 2′OMe modifications, whichcan be located throughout the molecule. In some embodiments, thenucleotide in position one of the guide strand (the nucleotide in themost 5′ position of the guide strand) is 2′OMe modified and/orphosphorylated. C and U nucleotides within the guide strand can be 2′Fmodified. For example, C and U nucleotides in positions 2-10 of a 19 ntguide strand (or corresponding positions in a guide strand of adifferent length) can be 2′F modified. C and U nucleotides within theguide strand can also be 2′OMe modified. For example, C and Unucleotides in positions 11-18 of a 19 nt guide strand (or correspondingpositions in a guide strand of a different length) can be 2′OMemodified. In some embodiments, the nucleotide at the most 3′ end of theguide strand is unmodified. In certain embodiments, the majority of Csand Us within the guide strand are 2′F modified and the 5′ end of theguide strand is phosphorylated. In other embodiments, position 1 and theCs or Us in positions 11-18 are 2′OMe modified and the 5′ end of theguide strand is phosphorylated. In other embodiments, position 1 and theCs or Us in positions 11-18 are 2′OMe modified, the 5′ end of the guidestrand is phosphorylated, and the Cs or Us in position 2-10 are 2′Fmodified.

In some aspects, an optimal passenger strand is approximately 11-14nucleotides in length. The passenger strand may contain modificationsthat confer increased stability. One or more nucleotides in thepassenger strand can be 2′OMe modified. In some embodiments, one or moreof the C and/or U nucleotides in the passenger strand is 2′OMe modified,or all of the C and U nucleotides in the passenger strand are 2′OMemodified. In certain embodiments, all of the nucleotides in thepassenger strand are 2′OMe modified. One or more of the nucleotides onthe passenger strand can also be phosphate-modified such asphosphorothioate modified. The passenger strand can also contain 2′ribo, 2′F and 2 deoxy modifications or any combination of the above.Chemical modification patterns on both the guide and passenger strandcan be well tolerated and a combination of chemical modifications canlead to increased efficacy and self-delivery of RNA molecules.

Aspects of the invention relate to RNAi constructs that have extendedsingle-stranded regions relative to double stranded regions, as comparedto molecules that have been used previously for RNAi. The singlestranded region of the molecules may be modified to promote cellularuptake or gene silencing. In some embodiments, phosphorothioatemodification of the single stranded region influences cellular uptakeand/or gene silencing. The region of the guide strand that isphosphorothioate modified can include nucleotides within both the singlestranded and double stranded regions of the molecule. In someembodiments, the single stranded region includes 2-12 phosphorothioatemodifications. For example, the single stranded region can include 2, 3,4, 5, 6, 7, 8, 9, 10, 11, or 12 phosphorothioate modifications. In someinstances, the single stranded region contains 6-8 phosphorothioatemodifications.

Molecules associated with the invention are also optimized for cellularuptake. In RNA molecules described herein, the guide and/or passengerstrands can be attached to a conjugate. In certain embodiments theconjugate is hydrophobic. The hydrophobic conjugate can be a smallmolecule with a partition coefficient that is higher than 10. Theconjugate can be a sterol-type molecule such as cholesterol, or amolecule with an increased length polycarbon chain attached to C17, andthe presence of a conjugate can influence the ability of an RNA moleculeto be taken into a cell with or without a lipid transfection reagent.The conjugate can be attached to the passenger or guide strand through ahydrophobic linker. In some embodiments, a hydrophobic linker is 5-12 Cin length, and/or is hydroxypyrrolidine-based. In some embodiments, ahydrophobic conjugate is attached to the passenger strand and the CUresidues of either the passenger and/or guide strand are modified. Insome embodiments, at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%or 95% of the CU residues on the passenger strand and/or the guidestrand are modified. In some aspects, molecules associated with theinvention are self-delivering (sd). As used herein, “self-delivery”refers to the ability of a molecule to be delivered into a cell withoutthe need for an additional delivery vehicle such as a transfectionreagent.

Aspects of the invention relate to selecting molecules for use in RNAi.In some embodiments, molecules that have a double stranded region of8-15 nucleotides can be selected for use in RNAi. In some embodiments,molecules are selected based on their thermodynamic stability (ΔG). Insome embodiments, molecules will be selected that have a (ΔG) of lessthan −13 kkal/mol. For example, the (ΔG) value may be −13, −14, −15,−16, −17, −18, −19, −21, −22 or less than −22 kkal/mol. In otherembodiments, the (ΔG) value may be higher than −13 kkal/mol. Forexample, the (ΔG) value may be −12, −11, −10, −9, −8, −7 or more than −7kkal/mol. It should be appreciated that AG can be calculated using anymethod known in the art. In some embodiments AG is calculated usingMfold, available through the Mfold internet site(mfold.bioinfo.rpi.edu/cgi-bin/rna-form1.cgi). Methods for calculatingΔG are described in, and are incorporated by reference from, thefollowing references: Zuker, M. (2003) Nucleic Acids Res.,31(13):3406-15; Mathews, D. H., Sabina, J., Zuker, M. and Turner, D. H.(1999) J. Mol. Biol. 288:911-940; Mathews, D. H., Disney, M. D., Childs,J. L., Schroeder, S. J., Zuker, M., and Turner, D. H. (2004) Proc. Natl.Acad. Sci. 101:7287-7292; Duan, S., Mathews, D. H., and Turner, D. H.(2006) Biochemistry 45:9819-9832; Wuchty, S., Fontana, W., Hofacker, I.L., and Schuster, P. (1999) Biopolymers 49:145-165.

In certain embodiments, the polynucleotide contains 5′- and/or 3′-endoverhangs. The number and/or sequence of nucleotides overhang on one endof the polynucleotide may be the same or different from the other end ofthe polynucleotide. In certain embodiments, one or more of the overhangnucleotides may contain chemical modification(s), such asphosphorothioate or 2′-OMe modification.

In certain embodiments, the polynucleotide is unmodified. In otherembodiments, at least one nucleotide is modified. In furtherembodiments, the modification includes a 2′-H or 2′-modified ribosesugar at the 2nd nucleotide from the 5′-end of the guide sequence. The“2nd nucleotide” is defined as the second nucleotide from the 5′-end ofthe polynucleotide.

As used herein, “2′-modified ribose sugar” includes those ribose sugarsthat do not have a 2′—OH group. “2′-modified ribose sugar” does notinclude 2′-deoxyribose (found in unmodified canonical DNA nucleotides).For example, the 2′-modified ribose sugar may be 2′-O-alkyl nucleotides,2′-deoxy-2′-fluoro nucleotides, 2′-deoxy nucleotides, or combinationthereof.

In certain embodiments, the 2′-modified nucleotides are pyrimidinenucleotides (e.g., C/U). Examples of 2′-O-alkyl nucleotides include2′-O-methyl nucleotides, or 2′-O-allyl nucleotides.

In certain embodiments, the sd-rxRNA polynucleotide of the inventionwith the above-referenced 5′-end modification exhibits significantly(e.g., at least about 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%,75%, 80%, 85%, 90% or more) less “off-target” gene silencing whencompared to similar constructs without the specified 5′-endmodification, thus greatly improving the overall specificity of the RNAireagent or therapeutics.

As used herein, “off-target” gene silencing refers to unintended genesilencing due to, for example, spurious sequence homology between theantisense (guide) sequence and the unintended target mRNA sequence.

According to this aspect of the invention, certain guide strandmodifications further increase nuclease stability, and/or lowerinterferon induction, without significantly decreasing RNAi activity (orno decrease in RNAi activity at all).

Certain combinations of modifications may result in further unexpectedadvantages, as partly manifested by enhanced ability to inhibit targetgene expression, enhanced serum stability, and/or increased targetspecificity, etc.

In certain embodiments, the guide strand comprises a 2′-O-methylmodified nucleotide at the 2^(nd) nucleotide on the 5′-end of the guidestrand and no other modified nucleotides.

In other aspects, the sd-rxRNA structures of the present inventionmediates sequence-dependent gene silencing by a microRNA mechanism. Asused herein, the term “microRNA” (“miRNA”), also referred to in the artas “small temporal RNAs” (“stRNAs”), refers to a small (10-50nucleotide) RNA which are genetically encoded (e.g., by viral,mammalian, or plant genomes) and are capable of directing or mediatingRNA silencing. An “miRNA disorder” shall refer to a disease or disordercharacterized by an aberrant expression or activity of an miRNA.

microRNAs are involved in down-regulating target genes in criticalpathways, such as development and cancer, in mice, worms and mammals.Gene silencing through a microRNA mechanism is achieved by specific yetimperfect base-pairing of the miRNA and its target messenger RNA (mRNA).Various mechanisms may be used in microRNA-mediated down-regulation oftarget mRNA expression.

miRNAs are noncoding RNAs of approximately 22 nucleotides which canregulate gene expression at the post transcriptional or translationallevel during plant and animal development. One common feature of miRNAsis that they are all excised from an approximately 70 nucleotideprecursor RNA stem-loop termed pre-miRNA, probably by Dicer, an RNaseIII-type enzyme, or a homolog thereof. Naturally-occurring miRNAs areexpressed by endogenous genes in vivo and are processed from a hairpinor stem-loop precursor (pre-miRNA or pri-miRNAs) by Dicer or otherRNAses. miRNAs can exist transiently in vivo as a double-stranded duplexbut only one strand is taken up by the RISC complex to direct genesilencing.

In some embodiments a version of sd-rxRNA compounds, which are effectivein cellular uptake and inhibiting of miRNA activity are described.Essentially the compounds are similar to RISC entering version but largestrand chemical modification patterns are optimized in the way to blockcleavage and act as an effective inhibitor of the RISC action.

For example, the compound might be completely or mostly O-methylmodified with the phosphorothioate content described previously. Forthese types of compounds the 5′ phosphorylation is not necessary in someembodiments. The presence of double stranded region is preferred as itis promotes cellular uptake and efficient RISC loading.

Another pathway that uses small RNAs as sequence-specific regulators isthe RNA interference (RNAi) pathway, which is an evolutionarilyconserved response to the presence of double-stranded RNA (dsRNA) in thecell. The dsRNAs are cleaved into ˜20-base pair (bp) duplexes ofsmall-interfering RNAs (siRNAs) by Dicer. These small RNAs get assembledinto multiprotein effector complexes called RNA-induced silencingcomplexes (RISCs). The siRNAs then guide the cleavage of target mRNAswith perfect complementarity.

Some aspects of biogenesis, protein complexes, and function are sharedbetween the siRNA pathway and the miRNA pathway. Single-strandedpolynucleotides may mimic the dsRNA in the siRNA mechanism, or themicroRNA in the miRNA mechanism.

In certain embodiments, the modified RNAi constructs may have improvedstability in serum and/or cerebral spinal fluid compared to anunmodified RNAi constructs having the same sequence.

In certain embodiments, the structure of the RNAi construct does notinduce interferon response in primary cells, such as mammalian primarycells, including primary cells from human, mouse and other rodents, andother non-human mammals. In certain embodiments, the RNAi construct mayalso be used to inhibit expression of a target gene in an invertebrateorganism.

To further increase the stability of the subject constructs in vivo, the3′-end of the structure may be blocked by protective group(s). Forexample, protective groups such as inverted nucleotides, inverted abasicmoieties, or amino-end modified nucleotides may be used. Invertednucleotides may comprise an inverted deoxynucleotide. Inverted abasicmoieties may comprise an inverted deoxyabasic moiety, such as a3′,3′-linked or 5′,5′-linked deoxyabasic moiety.

The RNAi constructs of the invention are capable of inhibiting thesynthesis of any target protein encoded by target gene(s). The inventionincludes methods to inhibit expression of a target gene either in a cellin vitro, or in vivo. As such, the RNAi constructs of the invention areuseful for treating a patient with a disease characterized by theoverexpression of a target gene.

The target gene can be endogenous or exogenous (e.g., introduced into acell by a virus or using recombinant DNA technology) to a cell. Suchmethods may include introduction of RNA into a cell in an amountsufficient to inhibit expression of the target gene. By way of example,such an RNA molecule may have a guide strand that is complementary tothe nucleotide sequence of the target gene, such that the compositioninhibits expression of the target gene.

The invention also relates to vectors expressing the nucleic acids ofthe invention, and cells comprising such vectors or the nucleic acids.The cell may be a mammalian cell in vivo or in culture, such as a humancell.

The invention further relates to compositions comprising the subjectRNAi constructs, and a pharmaceutically acceptable carrier or diluent.

The method may be carried out in vitro, ex vivo, or in vivo, in, forexample, mammalian cells in culture, such as a human cell in culture.

The target cells (e.g., mammalian cell) may be contacted in the presenceof a delivery reagent, such as a lipid (e.g., a cationic lipid) or aliposome.

Another aspect of the invention provides a method for inhibiting theexpression of a target gene in a mammalian cell, comprising contactingthe mammalian cell with a vector expressing the subject RNAi constructs.

In one aspect of the invention, a longer duplex polynucleotide isprovided, including a first polynucleotide that ranges in size fromabout 16 to about 30 nucleotides; a second polynucleotide that ranges insize from about 26 to about 46 nucleotides, wherein the firstpolynucleotide (the antisense strand) is complementary to both thesecond polynucleotide (the sense strand) and a target gene, and whereinboth polynucleotides form a duplex and wherein the first polynucleotidecontains a single stranded region longer than 6 bases in length and ismodified with alternative chemical modification pattern, and/or includesa conjugate moiety that facilitates cellular delivery. In thisembodiment, between about 40% to about 90% of the nucleotides of thepassenger strand between about 40% to about 90% of the nucleotides ofthe guide strand, and between about 40% to about 90% of the nucleotidesof the single stranded region of the first polynucleotide are chemicallymodified nucleotides.

In an embodiment, the chemically modified nucleotide in thepolynucleotide duplex may be any chemically modified nucleotide known inthe art, such as those discussed in detail above. In a particularembodiment, the chemically modified nucleotide is selected from thegroup consisting of 2′ F modified nucleotides, 2′-O-methyl modified and2′deoxy nucleotides. In another particular embodiment, the chemicallymodified nucleotides results from “hydrophobic modifications” of thenucleotide base. In another particular embodiment, the chemicallymodified nucleotides are phosphorothioates. In an additional particularembodiment, chemically modified nucleotides are combination ofphosphorothioates, 2′-O-methyl, 2′deoxy, hydrophobic modifications andphosphorothioates. As these groups of modifications refer tomodification of the ribose ring, back bone and nucleotide, it isfeasible that some modified nucleotides will carry a combination of allthree modification types.

In another embodiment, the chemical modification is not the same acrossthe various regions of the duplex. In a particular embodiment, the firstpolynucleotide (the passenger strand), has a large number of diversechemical modifications in various positions. For this polynucleotide upto 90% of nucleotides might be chemically modified and/or havemismatches introduced.

In another embodiment, chemical modifications of the first or secondpolynucleotide include, but not limited to, 5′ position modification ofUridine and Cytosine (4-pyridyl, 2-pyridyl, indolyl, phenyl (C₆H₅OH);tryptophanyl (C8H6N)CH2CH(NH2)CO), isobutyl, butyl, aminobenzyl; phenyl;naphthyl, etc.), where the chemical modification might alter basepairing capabilities of a nucleotide. For the guide strand an importantfeature of this aspect of the invention is the position of the chemicalmodification relative to the 5′ end of the antisense and sequence. Forexample, chemical phosphorylation of the 5′ end of the guide strand isusually beneficial for efficacy. O-methyl modifications in the seedregion of the sense strand (position 2-7 relative to the 5′ end) are notgenerally well tolerated, whereas 2′F and deoxy are well tolerated. Themid part of the guide strand and the 3′ end of the guide strand are morepermissive in a type of chemical modifications applied. Deoxymodifications are not tolerated at the 3′ end of the guide strand.

A unique feature of this aspect of the invention involves the use ofhydrophobic modification on the bases. In one embodiment, thehydrophobic modifications are preferably positioned near the 5′ end ofthe guide strand, in other embodiments, they localized in the middle ofthe guides strand, in other embodiment they localized at the 3′ end ofthe guide strand and yet in another embodiment they are distributedthought the whole length of the polynucleotide. The same type ofpatterns is applicable to the passenger strand of the duplex.

The other part of the molecule is a single stranded region. In someembodiments, the single stranded region is expected to range from 7 to40 nucleotides.

In one embodiment, the single stranded region of the firstpolynucleotide contains modifications selected from the group consistingof between 40% and 90% hydrophobic base modifications, between 40%-90%phosphorothioates, between 40%-90% modification of the ribose moiety,and any combination of the preceding.

Efficiency of guide strand (first polynucleotide) loading into the RISCcomplex might be altered for heavily modified polynucleotides, so in oneembodiment, the duplex polynucleotide includes a mismatch betweennucleotide 9, 11, 12, 13, or 14 on the guide strand (firstpolynucleotide) and the opposite nucleotide on the sense strand (secondpolynucleotide) to promote efficient guide strand loading.

More detailed aspects of the invention are described in the sectionsbelow.

Duple Characteristics

Double-stranded oligonucleotides of the invention may be formed by twoseparate complementary nucleic acid strands. Duplex formation can occureither inside or outside the cell containing the target gene.

As used herein, the term “duplex” includes the region of thedouble-stranded nucleic acid molecule(s) that is (are) hydrogen bondedto a complementary sequence. Double-stranded oligonucleotides of theinvention may comprise a nucleotide sequence that is sense to a targetgene and a complementary sequence that is antisense to the target gene.The sense and antisense nucleotide sequences correspond to the targetgene sequence, e.g., are identical or are sufficiently identical toeffect target gene inhibition (e.g., are about at least about 98%identical, 96% identical, 94%, 90% identical, 85% identical, or 80%identical) to the target gene sequence.

In certain embodiments, the double-stranded oligonucleotide of theinvention is double-stranded over its entire length, i.e., with nooverhanging single-stranded sequence at either end of the molecule,i.e., is blunt-ended. In other embodiments, the individual nucleic acidmolecules can be of different lengths. In other words, a double-strandedoligonucleotide of the invention is not double-stranded over its entirelength. For instance, when two separate nucleic acid molecules are used,one of the molecules, e.g., the first molecule comprising an antisensesequence, can be longer than the second molecule hybridizing thereto(leaving a portion of the molecule single-stranded). Likewise, when asingle nucleic acid molecule is used a portion of the molecule at eitherend can remain single-stranded.

In one embodiment, a double-stranded oligonucleotide of the inventioncontains mismatches and/or loops or bulges, but is double-stranded overat least about 70% of the length of the oligonucleotide. In anotherembodiment, a double-stranded oligonucleotide of the invention isdouble-stranded over at least about 80% of the length of theoligonucleotide. In another embodiment, a double-strandedoligonucleotide of the invention is double-stranded over at least about90%-95% of the length of the oligonucleotide. In another embodiment, adouble-stranded oligonucleotide of the invention is double-stranded overat least about 96%-98% of the length of the oligonucleotide. In certainembodiments, the double-stranded oligonucleotide of the inventioncontains at least or up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,14, or 15 mismatches.

Modifications

The nucleotides of the invention may be modified at various locations,including the sugar moiety, the phosphodiester linkage, and/or the base.

In some embodiments, the base moiety of a nucleoside may be modified.For example, a pyrimidine base may be modified at the 2, 3, 4, 5, and/or6 position of the pyrimidine ring. In some embodiments, the exocyclicamine of cytosine may be modified. A purine base may also be modified.For example, a purine base may be modified at the 1, 2, 3, 6, 7, or 8position. In some embodiments, the exocyclic amine of adenine may bemodified. In some cases, a nitrogen atom in a ring of a base moiety maybe substituted with another atom, such as carbon. A modification to abase moiety may be any suitable modification. Examples of modificationsare known to those of ordinary skill in the art. In some embodiments,the base modifications include alkylated purines or pyrimidines,acylated purines or pyrimidines, or other heterocycles.

In some embodiments, a pyrimidine may be modified at the 5 position. Forexample, the 5 position of a pyrimidine may be modified with an alkylgroup, an alkynyl group, an alkenyl group, an acyl group, or substitutedderivatives thereof. In other examples, the 5 position of a pyrimidinemay be modified with a hydroxyl group or an alkoxyl group or substitutedderivative thereof. Also, the N⁴ position of a pyrimidine may bealkylated. In still further examples, the pyrimidine 5-6 bond may besaturated, a nitrogen atom within the pyrimidine ring may be substitutedwith a carbon atom, and/or the O² and O⁴ atoms may be substituted withsulfur atoms. It should be understood that other modifications arepossible as well.

In other examples, the N⁷ position and/or N² and/or N³ position of apurine may be modified with an alkyl group or substituted derivativethereof. In further examples, a third ring may be fused to the purinebicyclic ring system and/or a nitrogen atom within the purine ringsystem may be substituted with a carbon atom. It should be understoodthat other modifications are possible as well.

Non-limiting examples of pyrimidines modified at the 5 position aredisclosed in U.S. Pat. No. 5,591,843, U.S. Pat. No. 7,205,297, U.S. Pat.No. 6,432,963, and U.S. Pat. No. 6,020,483; non-limiting examples ofpyrimidines modified at the N⁴ position are disclosed in U.S. Pat. No.5,580,731; non-limiting examples of purines modified at the 8 positionare disclosed in U.S. Pat. No. 6,355,787 and U.S. Pat. No. 5,580,972;non-limiting examples of purines modified at the N⁶ position aredisclosed in U.S. Pat. No. 4,853,386, U.S. Pat. No. 5,789,416, and U.S.Pat. No. 7,041,824; and non-limiting examples of purines modified at the2 position are disclosed in U.S. Pat. No. 4,201,860 and U.S. Pat. No.5,587,469, all of which are incorporated herein by reference.

Non-limiting examples of modified bases include N⁴,N⁴-ethanocytosine,7-deazaxanthosine, 7-deazaguanosine, 8-oxo-, N⁶-methyladenine,4-acetylcytosine, 5-(carboxyhydroxylmethyl) uracil, 5-fluorouracil,5-bromouracil, 5-carboxymethylaminomethyl-2-thiouracil,5-carboxymethylaminomethyl uracil, dihydrouracil, inosine,N⁶-isopentenyl-adenine, 1-methyladenine, 1-methylpseudouracil,1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine,2-methylguanine, 3-methylcytosine, 5-methylcytosine, N⁶-methyladenine,7-methylguanine, 5-methylaminomethyl uracil, 5-methoxyaminomethyl-2-thiouracil, 5-methoxyuracil,2-methylthio-N⁶-isopentenyladenine, pseudouracil, 5-methyl-2-thiouracil,2-thiouracil, 4-thiouracil, 5-methyluracil, 2-thiocytosine, and2,6-diaminopurine. In some embodiments, the base moiety may be aheterocyclic base other than a purine or pyrimidine. The heterocyclicbase may be optionally modified and/or substituted.

Sugar moieties include natural, unmodified sugars, e.g., monosaccharide(such as pentose, e.g., ribose, deoxyribose), modified sugars and sugaranalogs. In general, possible modifications of nucleomonomers,particularly of a sugar moiety, include, for example, replacement of oneor more of the hydroxyl groups with a halogen, a heteroatom, analiphatic group, or the functionalization of the hydroxyl group as anether, an amine, a thiol, or the like.

One particularly useful group of modified nucleomonomers are 2′-O-methylnucleotides. Such 2′-O-methyl nucleotides may be referred to as“methylated,” and the corresponding nucleotides may be made fromunmethylated nucleotides followed by alkylation or directly frommethylated nucleotide reagents. Modified nucleomonomers may be used incombination with unmodified nucleomonomers. For example, anoligonucleotide of the invention may contain both methylated andunmethylated nucleomonomers.

Some exemplary modified nucleomonomers include sugar- orbackbone-modified ribonucleotides. Modified ribonucleotides may containa non-naturally occurring base (instead of a naturally occurring base),such as uridines or cytidines modified at the 5′-position, e.g.,5′-(2-amino)propyl uridine and 5′-bromo uridine; adenosines andguanosines modified at the 8-position, e.g., 8-bromo guanosine; deazanucleotides, e.g., 7-deaza-adenosine; and N-alkylated nucleotides, e.g.,N6-methyl adenosine. Also, sugar-modified ribonucleotides may have the2′—OH group replaced by a H, alxoxy (or OR), R or alkyl, halogen, SH,SR, amino (such as NH₂, NHR, NR₂), or CN group, wherein R is loweralkyl, alkenyl, or alkynyl.

Modified ribonucleotides may also have the phosphodiester groupconnecting to adjacent ribonucleotides replaced by a modified group,e.g., of phosphorothioate group. More generally, the various nucleotidemodifications may be combined.

Although the antisense (guide) strand may be substantially identical toat least a portion of the target gene (or genes), at least with respectto the base pairing properties, the sequence need not be perfectlyidentical to be useful, e.g., to inhibit expression of a target gene'sphenotype. Generally, higher homology can be used to compensate for theuse of a shorter antisense gene. In some cases, the antisense strandgenerally will be substantially identical (although in antisenseorientation) to the target gene.

The use of 2′-O-methyl modified RNA may also be beneficial incircumstances in which it is desirable to minimize cellular stressresponses. RNA having 2′-O-methyl nucleomonomers may not be recognizedby cellular machinery that is thought to recognize unmodified RNA. Theuse of 2′-O-methylated or partially 2′-O-methylated RNA may avoid theinterferon response to double-stranded nucleic acids, while maintainingtarget RNA inhibition. This may be useful, for example, for avoiding theinterferon or other cellular stress responses, both in short RNAi (e.g.,siRNA) sequences that induce the interferon response, and in longer RNAisequences that may induce the interferon response.

Overall, modified sugars may include D-ribose, 2′-O-alkyl (including2′-O-methyl and 2′-O-ethyl), i.e., 2′-alkoxy, 2′-amino, 2′-S-alkyl,2′-halo (including 2′-fluoro), 2′-methoxyethoxy, 2′-allyloxy(—OCH₂CH═CH₂), 2′-propargyl, 2′-propyl, ethynyl, ethenyl, propenyl, andcyano and the like. In one embodiment, the sugar moiety can be a hexoseand incorporated into an oligonucleotide as described (Augustyns, K., etal., Nucl. Acids. Res. 18:4711 (1992)). Exemplary nucleomonomers can befound, e.g., in U.S. Pat. No. 5,849,902, incorporated by referenceherein.

Definitions of specific functional groups and chemical terms aredescribed in more detail below. For purposes of this invention, thechemical elements are identified in accordance with the Periodic Tableof the Elements, CAS version, Handbook of Chemistry and Physics, 75^(th)Ed., inside cover, and specific functional groups are generally definedas described therein. Additionally, general principles of organicchemistry, as well as specific functional moieties and reactivity, aredescribed in Organic Chemistry, Thomas Sorrell, University ScienceBooks, Sausalito: 1999, the entire contents of which are incorporatedherein by reference.

Certain compounds of the present invention may exist in particulargeometric or stereoisomeric forms. The present invention contemplatesall such compounds, including cis- and trans-isomers, R- andS-enantiomers, diastereomers, (D)-isomers, (L)-isomers, the racemicmixtures thereof, and other mixtures thereof, as falling within thescope of the invention. Additional asymmetric carbon atoms may bepresent in a substituent such as an alkyl group. All such isomers, aswell as mixtures thereof, are intended to be included in this invention.

Isomeric mixtures containing any of a variety of isomer ratios may beutilized in accordance with the present invention. For example, whereonly two isomers are combined, mixtures containing 50:50, 60:40, 70:30,80:20, 90:10, 95:5, 96:4, 97:3, 98:2, 99:1, or 100:0 isomer ratios areall contemplated by the present invention. Those of ordinary skill inthe art will readily appreciate that analogous ratios are contemplatedfor more complex isomer mixtures.

If, for instance, a particular enantiomer of a compound of the presentinvention is desired, it may be prepared by asymmetric synthesis, or byderivation with a chiral auxiliary, where the resulting diastereomericmixture is separated and the auxiliary group cleaved to provide the puredesired enantiomers. Alternatively, where the molecule contains a basicfunctional group, such as amino, or an acidic functional group, such ascarboxyl, diastereomeric salts are formed with an appropriateoptically-active acid or base, followed by resolution of thediastereomers thus formed by fractional crystallization orchromatographic means well known in the art, and subsequent recovery ofthe pure enantiomers.

In certain embodiments, oligonucleotides of the invention comprise 3′and 5′ termini (except for circular oligonucleotides). In oneembodiment, the 3′ and 5′ termini of an oligonucleotide can besubstantially protected from nucleases e.g., by modifying the 3′ or 5′linkages (e.g., U.S. Pat. No. 5,849,902 and WO 98/13526). For example,oligonucleotides can be made resistant by the inclusion of a “blockinggroup.” The term “blocking group” as used herein refers to substituents(e.g., other than OH groups) that can be attached to oligonucleotides ornucleomonomers, either as protecting groups or coupling groups forsynthesis (e.g., FITC, propyl (CH₂—CH₂—CH₃), glycol (—O—CH₂—CH₂—O—)phosphate (PO₃ ²⁻), hydrogen phosphonate, or phosphoramidite). “Blockinggroups” also include “end blocking groups” or “exonuclease blockinggroups” which protect the 5′ and 3′ termini of the oligonucleotide,including modified nucleotides and non-nucleotide exonuclease resistantstructures.

Exemplary end-blocking groups include cap structures (e.g., a7-methylguanosine cap), inverted nucleomonomers, e.g., with 3′→3′ or5′→5′ end inversions (see, e.g., Ortiagao et al. 1992. Antisense Res.Dev. 2:129), methylphosphonate, phosphoramidite, non-nucleotide groups(e.g., non-nucleotide linkers, amino linkers, conjugates) and the like.The 3′ terminal nucleomonomer can comprise a modified sugar moiety. The3′ terminal nucleomonomer comprises a 3′-O that can optionally besubstituted by a blocking group that prevents 3′-exonuclease degradationof the oligonucleotide. For example, the 3′-hydroxyl can be esterifiedto a nucleotide through a 3′→3′ internucleotide linkage. For example,the alkyloxy radical can be methoxy, ethoxy, or isopropoxy, andpreferably, ethoxy. Optionally, the 3′→3′linked nucleotide at the 3′terminus can be linked by a substitute linkage. To reduce nucleasedegradation, the 5′ most 3′→5′ linkage can be a modified linkage, e.g.,a phosphorothioate or a P-alkyloxyphosphotriester linkage. Preferably,the two 5′ most 3′→5′ linkages are modified linkages. Optionally, the 5′terminal hydroxy moiety can be esterified with a phosphorus containingmoiety, e.g., phosphate, phosphorothioate, or P-ethoxyphosphate.

One of ordinary skill in the art will appreciate that the syntheticmethods, as described herein, utilize a variety of protecting groups. Bythe term “protecting group,” as used herein, it is meant that aparticular functional moiety, e.g., O, S, or N, is temporarily blockedso that a reaction can be carried out selectively at another reactivesite in a multifunctional compound. In certain embodiments, a protectinggroup reacts selectively in good yield to give a protected substratethat is stable to the projected reactions: the protecting group shouldbe selectively removable in good yield by readily available, preferablynon-toxic reagents that do not attack the other functional groups; theprotecting group forms an easily separable derivative (more preferablywithout the generation of new stereogenic centers): and the protectinggroup has a minimum of additional functionality to avoid further sitesof reaction. As detailed herein, oxygen, sulfur, nitrogen, and carbonprotecting groups may be utilized. Hydroxyl protecting groups includemethyl, methoxylmethyl (MOM), methylthiomethyl (MTM), t-butylthiomethyl,(phenyldimethylsilyl)methoxymethyl (SMOM), benzyloxymethyl (BOM),p-methoxybenzyloxymethyl (PMBM), (4-methoxyphenoxy)methyl (p-AOM),guaiacolmethyl (GUM), 1-butoxymethyl, 4-pentenyloxymethyl (POM),siloxymethyl, 2-methoxyethoxymethyl (MEM), 2,2,2-trichloroethoxymethyl,bis(2-chloroethoxy)methyl, 2-(trimethylsilyl)ethoxymethyl (SEMOR),tetrahydropyranyl (THP), 3-bromotetrahydropyranyl,tetrahydrothiopyranyl, 1-methoxycyclohexyl, 4-methoxytetrahydropyranyl(MTHP), 4-methoxytetrahydrothiopyranyl, 4-methoxytetrahydrothiopyranylS,S-dioxide, 1-[(2-chloro-4-methyl)phenyl]-4-methoxypiperidin-4-yl(CTMP), 1,4-dioxan-2-yl, tetrahydrofuranyl, tetrahydrothiofuranyl,2,3,3a,4,5,6,7,7a-octahydro-7,8,8-trimethyl-4,7-methanobenzofuran-2-yl,1-ethoxyethyl, 1-(2-chloroethoxy)ethyl, 1-methyl-1-methoxyethyl,1-methyl-1-benzyloxyethyl, 1-methyl-1-benzyloxy-2-fluoroethyl,2,2,2-trichloroethyl, 2-trimethylsilylethyl, 2-(phenylselenyl)ethyl,t-butyl, allyl, p-chlorophenyl, p-methoxyphenyl, 2,4-dinitrophenyl,benzyl, p-methoxybenzyl, 3,4-dimethoxybenzyl, o-nitrobenzyl,p-nitrobenzyl, p-halobenzyl, 2,6-dichlorobenzyl, p-cyanobenzyl,p-phenylbenzyl, 2-picolyl, 4-picolyl, 3-methyl-2-picolyl N-oxido,diphenylmethyl, p,p′-dinitrobenzhydryl, 5-dibenzosuberyl,triphenylmethyl, a-naphthyldiphenylmethyl,p-methoxyphenyldiphenylmethyl, di(p-methoxyphenyl)phenylmethyl,tri(p-methoxyphenyl)methyl, 4-(4′-bromophenacyloxyphenyl)diphenylmethyl,4,4′,4″-tris(4,5-dichlorophthalimidophenyl)methyl,4,4′,4″-tris(levulinoyloxyphenyl)methyl,4,4′,4″-tris(benzoyloxyphenyl)methyl,3-(imidazol-1-yl)bis(4′,4″-dimethoxyphenyl)methyl,1,1-bis(4-methoxyphenyl)-1′-pyrenylmethyl, 9-anthryl,9-(9-phenyl)xanthenyl, 9-(9-phenyl-10-oxo)anthryl,1,3-benzodithiolan-2-yl, benzisothiazolyl S,S-dioxido, trimethylsilyl(TMS), triethylsilyl (TES), triisopropylsilyl (TIPS),dimethylisopropylsilyl (IPDMS), diethylisopropylsilyl (DEIPS),dimethylthexylsilyl, t-butyldimethylsilyl (TBDMS), t-butyldiphenylsilyl(TBDPS), tribenzylsilyl, tri-p-xylylsilyl, triphenylsilyl,diphenylmethylsilyl (DPMS), t-butylmethoxyphenylsilyl (TBMPS), formate,benzoylformate, acetate, chloroacetate, dichloroacetate,trichloroacetate, trifluoroacetate, methoxyacetate,triphenylmethoxyacetate, phenoxyacetate, p-chlorophenoxyacetate,3-phenylpropionate, 4-oxopentanoate (levulinate),4,4-(ethylenedithio)pentanoate (levulinoyldithioacetal), pivaloate,adamantoate, crotonate, 4-methoxycrotonate, benzoate, p-phenylbenzoate,2,4,6-trimethylbenzoate (mesitoate), alkyl methyl carbonate,9-fluorenylmethyl carbonate (Fmoc), alkyl ethyl carbonate, alkyl2,2,2-trichloroethyl carbonate (Troc), 2-(trimethylsilyl)ethyl carbonate(TMSEC), 2-(phenylsulfonyl) ethyl carbonate (Psec),2-(triphenylphosphonio) ethyl carbonate (Peoc), alkyl isobutylcarbonate, alkyl vinyl carbonate alkyl allyl carbonate, alkylp-nitrophenyl carbonate, alkyl benzyl carbonate, alkyl p-methoxybenzylcarbonate, alkyl 3,4-dimethoxybenzyl carbonate, alkyl o-nitrobenzylcarbonate, alkyl p-nitrobenzyl carbonate, alkyl S-benzyl thiocarbonate,4-ethoxy-1-naphthyl carbonate, methyl dithiocarbonate, 2-iodobenzoate,4-azidobutyrate, 4-nitro-4-methylpentanoate, o-(dibromomethyl)benzoate,2-formylbenzenesulfonate, 2-(methylthiomethoxy)ethyl,4-(methylthiomethoxy)butyrate, 2-(methylthiomethoxymethyl)benzoate,2,6-dichloro-4-methylphenoxyacetate,2,6-dichloro-4-(1,1,3,3-tetramethylbutyl)phenoxyacetate,2,4-bis(1,1-dimethylpropyl)phenoxyacetate, chlorodiphenylacetate,isobutyrate, monosuccinoate, (E)-2-methyl-2-butenoate,o-(methoxycarbonyl)benzoate, a-naphthoate, nitrate, alkylN,N,N′,N′-tetramethylphosphorodiamidate, alkyl N-phenylcarbamate,borate, dimethylphosphinothioyl, alkyl 2,4-dinitrophenylsulfenate,sulfate, methanesulfonate (mesylate), benzylsulfonate, and tosylate(Ts). For protecting 1,2- or 1,3-diols, the protecting groups includemethylene acetal, ethylidene acetal, 1-t-butylethylidene ketal,1-phenylethylidene ketal, (4-methoxyphenyl)ethylidene acetal,2,2,2-trichloroethylidene acetal, acetonide, cyclopentylidene ketal,cyclohexylidene ketal, cycloheptylidene ketal, benzylidene acetal,p-methoxybenzylidene acetal, 2,4-dimethoxybenzylidene ketal,3,4-dimethoxybenzylidene acetal, 2-nitrobenzylidene acetal,methoxymethylene acetal, ethoxymethylene acetal, dimethoxymethyleneortho ester, 1-methoxyethylidene ortho ester, 1-ethoxyethylidine orthoester, 1,2-dimethoxyethylidene ortho ester, α-methoxybenzylidene orthoester, 1-(N,N-dimethylamino)ethylidene derivative,α-(N,N′-dimethylamino)benzylidene derivative, 2-oxacyclopentylideneortho ester, di-t-butylsilylene group (DTBS),1,3-(1,1,3,3-tetraisopropyldisiloxanylidene) derivative (TIPDS),tetra-t-butoxydisiloxane-1,3-diylidene derivative (TBDS), cycliccarbonates, cyclic boronates, ethyl boronate, and phenyl boronate.Amino-protecting groups include methyl carbamate, ethyl carbamante,9-fluorenylmethyl carbamate (Fmoc), 9-(2-sulfo)fluorenylmethylcarbamate, 9-(2,7-dibromo)fluorenylmethyl carbamate,2,7-di-t-butyl-[9-(10,10-dioxo-10,10,10,10-tetrahydrothioxanthyl)]methylcarbamate (DBD-Tmoc), 4-methoxyphenacyl carbamate (Phenoc),2,2,2-trichloroethyl carbamate (Troc), 2-trimethylsilylethyl carbamate(Teoc), 2-phenylethyl carbamate (hZ), 1-(1-adamantyl)-1-methylethylcarbamate (Adpoc), 1,1-dimethyl-2-haloethyl carbamate,1,1-dimethyl-2,2-dibromoethyl carbamate (DB-t-BOC),1,1-dimethyl-2,2,2-trichloroethyl carbamate (TCBOC),1-methyl-1-(4-biphenylyl)ethyl carbamate (Bpoc),1-(3,5-di-t-butylphenyl)-1-methylethyl carbamate (t-Bumeoc), 2-(2′- and4′-pyridyl)ethyl carbamate (Pyoc), 2-(NN-dicyclohexylcarboxamido)ethylcarbamate, t-butyl carbamate (BOC), 1-adamantyl carbamate (Adoc), vinylcarbamate (Voc), allyl carbamate (Alloc), 1-isopropylallyl carbamate(Ipaoc), cinnamyl carbamate (Coc), 4-nitrocinnamyl carbamate (Noc),8-quinolyl carbamate, N-hydroxypiperidinyl carbamate, alkyldithiocarbamate, benzyl carbamate (Cbz), p-methoxybenzyl carbamate (Moz),p-nitobenzyl carbamate, p-bromobenzyl carbamate, p-chlorobenzylcarbamate, 2,4-dichlorobenzyl carbamate, 4-methylsulfinylbenzylcarbamate (Msz), 9-anthrylmethyl carbamate, diphenylmethyl carbamate,2-methylthioethyl carbamate, 2-methylsulfonylethyl carbamate,2-(p-toluenesulfonyl)ethyl carbamate, [2-(1,3-dithianyl)]methylcarbamate (Dmoc), 4-methylthiophenyl carbamate (Mtpc),2,4-dimethylthiophenyl carbamate (Bmpc), 2-phosphonioethyl carbamate(Peoc), 2-triphenylphosphonioisopropyl carbamate (Ppoc),1,1-dimethyl-2-cyanoethyl carbamate, m-chloro-p-acyloxybenzyl carbamate,p-(dihydroxyboryl)benzyl carbamate, 5-benzisoxazolylmethyl carbamate,2-(trifluoromethyl)-6-chromonylmethyl carbamate (Tcroc), m-nitrophenylcarbamate, 3,5-dimethoxybenzyl carbamate, o-nitrobenzyl carbamate,3,4-dimethoxy-6-nitrobenzyl carbamate, phenyl(o-nitrophenyl)methylcarbamate, phenothiazinyl-(10)-carbonyl derivative,N′-p-toluenesulfonylaminocarbonyl derivative, N′-phenylaminothiocarbonylderivative, t-amyl carbamate, S-benzyl thiocarbamate, p-cyanobenzylcarbamate, cyclobutyl carbamate, cyclohexyl carbamate, cyclopentylcarbamate, cyclopropylmethyl carbamate, p-decyloxybenzyl carbamate,2,2-dimethoxycarbonylvinyl carbamate, o-(N,N-dimethylcarboxamido)benzylcarbamate, 1,1-dimethyl-3-(N,N-dimethylcarboxamido)propyl carbamate,1,1-dimethylpropynyl carbamate, di(2-pyridyl)methyl carbamate,2-furanylmethyl carbamate, 2-iodoethyl carbamate, isobornyl carbamate,isobutyl carbamate, isonicotinyl carbamate,p-(p′-methoxyphenylazo)benzyl carbamate, 1-methylcyclobutyl carbamate,1-methylcyclohexyl carbamate, 1-methyl-1-cyclopropylmethyl carbamate,1-methyl-1-(3,5-dimethoxyphenyl)ethyl carbamate,1-methyl-1-(p-phenylazophenyl)ethyl carbamate, 1-methyl-1-phenylethylcarbamate, 1-methyl-1-(4-pyridyl)ethyl carbamate, phenyl carbamate,p-(phenylazo)benzyl carbamate, 2,4,6-tri-t-butylphenyl carbamate,4-(trimethylammonium)benzyl carbamate, 2,4,6-trimethylbenzyl carbamate,formamide, acetamide, chloroacetamide, trichloroacetamide,trifluoroacetamide, phenylacetamide, 3-phenylpropanamide, picolinamide,3-pyridylcarboxamide, N-benzoylphenylalanyl derivative, benzamide,p-phenylbenzamide, o-nitophenylacetamide, o-nitrophenoxyacetamide,acetoacetamide, (N′-dithiobenzyloxycarbonylamino)acetamide,3-(p-hydroxyphenyl)propanamide, 3-(o-nitrophenyl)propanamide,2-methyl-2-(o-nitrophenoxy)propanamide,2-methyl-2-(o-phenylazophenoxy)propanamide, 4-chlorobutanamide,3-methyl-3-nitrobutanamide, o-nitrocinnamide, N-acetylmethioninederivative, o-nitrobenzamide, o-(benzoyloxymethyl)benzamide,4,5-diphenyl-3-oxazolin-2-one, N-phthalimide, N-dithiasuccinimide (Dts),N-2,3-diphenylmaleimide, N-2,5-dimethylpyrrole,N-1,1,4,4-tetramethyldisilylazacyclopentane adduct (STABASE),5-substituted 1,3-dimethyl-1,3,5-triazacyclohexan-2-one, 5-substituted1,3-dibenzyl-1,3,5-triazacyclohexan-2-one, 1-substituted3,5-dinitro-4-pyridone, N-methylamine, N-allylamine,N-[2-(trimethylsilyl)ethoxy]methylamine (SEM), N-3-acetoxypropylamine,N-(1-isopropyl-4-nitro-2-oxo-3-pyroolin-3-yl)amine, quaternary ammoniumsalts, N-benzylamine, N-di(4-methoxyphenyl)methylamine,N-5-dibenzosuberylamine, N-triphenylmethylamine (Tr),N-[(4-methoxyphenyl)diphenylmethyl]amine (MMTr),N-9-phenylfluorenylamine (PhF),N-2,7-dichloro-9-fluorenylmethyleneamine, N-ferrocenylmethylamino (Fcm),N-2-picolylamino N′-oxide, N-1,1-dimethylthiomethyleneamine,N-benzylideneamine, N-p-methoxybenzylideneamine,N-diphenylmethyleneamine, N-[(2-pyridyl)mesityl]methyleneamine,N—(N′,N′-dimethylaminomethylene)amine, N,N′-isopropylidenediamine,N-p-nitrobenzylideneamine, N-salicylideneamine,N-5-chlorosalicylideneamine,N-(5-chloro-2-hydroxyphenyl)phenylmethyleneamine,N-cyclohexylideneamine, N-(5,5-dimethyl-3-oxo-1-cyclohexenyl)amine,N-borane derivative, N-diphenylborinic acid derivative,N-[phenyl(pentacarbonylchromium- or tungsten)carbonyl]amine, N-copperchelate, N-zinc chelate, N-nitroamine, N-nitrosoamine, amine N-oxide,diphenylphosphinamide (Dpp), dimethylthiophosphinamide (Mpt),diphenylthiophosphinamide (Ppt), dialkyl phosphoramidates, dibenzylphosphoramidate, diphenyl phosphoramidate, benzenesulfenamide,o-nitrobenzenesulfenamide (Nps), 2,4-dinitrobenzenesulfenamide,pentachlorobenzenesulfenamide, 2-nitro-4-methoxybenzenesulfenamide,triphenylmethylsulfenamide, 3-nitropyridinesulfenamide (Npys),p-toluenesulfonamide (Ts), benzenesulfonamide,2,3,6,-trimethyl-4-methoxybenzenesulfonamide (Mtr),2,4,6-trimethoxybenzenesulfonamide (Mtb),2,6-dimethyl-4-methoxybenzenesulfonamide (Pinme),2,3,5,6-tetramethyl-4-methoxybenzenesulfonamide (Mte),4-methoxybenzenesulfonamide (Mbs), 2,4,6-trimethylbenzenesulfonamide(Mts), 2,6-dimethoxy-4-methylbenzenesulfonamide (iMds),2,2,5,7,8-pentamethylchroman-6-sulfonamide (Pmc), methanesulfonamide(Ms), β-trimethylsilylethanesulfonamide (SES), 9-anthracenesulfonamide,4-(4′,8′-dimethoxynaphthylmethyl)benzenesulfonamide (DNMBS),benzylsulfonamide, trifluoromethylsulfonamide, and phenacylsulfonamide.Exemplary protecting groups are detailed herein. However, it will beappreciated that the present invention is not intended to be limited tothese protecting groups; rather, a variety of additional equivalentprotecting groups can be readily identified using the above criteria andutilized in the method of the present invention. Additionally, a varietyof protecting groups are described in Protective Groups in OrganicSynthesis, Third Ed. Greene, T. W. and Wuts, P. G., Eds., John Wiley &Sons, New York: 1999, the entire contents of which are herebyincorporated by reference.

It will be appreciated that the compounds, as described herein, may besubstituted with any number of substituents or functional moieties. Ingeneral, the term “substituted” whether preceded by the term“optionally” or not, and substituents contained in formulas of thisinvention, refer to the replacement of hydrogen radicals in a givenstructure with the radical of a specified substituent. When more thanone position in any given structure may be substituted with more thanone substituent selected from a specified group, the substituent may beeither the same or different at every position. As used herein, the term“substituted” is contemplated to include all permissible substituents oforganic compounds. In a broad aspect, the permissible substituentsinclude acyclic and cyclic, branched and unbranched, carbocyclic andheterocyclic, aromatic and nonaromatic substituents of organiccompounds. Heteroatoms such as nitrogen may have hydrogen substituentsand/or any permissible substituents of organic compounds describedherein which satisfy the valencies of the heteroatoms. Furthermore, thisinvention is not intended to be limited in any manner by the permissiblesubstituents of organic compounds. Combinations of substituents andvariables envisioned by this invention are preferably those that resultin the formation of stable compounds useful in the treatment, forexample, of infectious diseases or proliferative disorders. The term“stable”, as used herein, preferably refers to compounds which possessstability sufficient to allow manufacture and which maintain theintegrity of the compound for a sufficient period of time to be detectedand preferably for a sufficient period of time to be useful for thepurposes detailed herein.

The term “aliphatic,” as used herein, includes both saturated andunsaturated, straight chain (i.e., unbranched), branched, acyclic,cyclic, or polycyclic aliphatic hydrocarbons, which are optionallysubstituted with one or more functional groups. As will be appreciatedby one of ordinary skill in the art, “aliphatic” is intended herein toinclude, but is not limited to, alkyl, alkenyl, alkynyl, cycloalkyl,cycloalkenyl, and cycloalkynyl moieties. Thus, as used herein, the term“alkyl” includes straight, branched and cyclic alkyl groups. Ananalogous convention applies to other generic terms such as “alkenyl,”“alkynyl,” and the like. Furthermore, as used herein, the terms “alkyl,”“alkenyl,” “alkynyl,” and the like encompass both substituted andunsubstituted groups. In certain embodiments, as used herein, “loweralkyl” is used to indicate those alkyl groups (cyclic, acyclic,substituted, unsubstituted, branched, or unbranched) having 1-6 carbonatoms.

In certain embodiments, the alkyl, alkenyl, and alkynyl groups employedin the invention contain 1-20 aliphatic carbon atoms. In certain otherembodiments, the alkyl, alkenyl, and alkynyl groups employed in theinvention contain 1-10 aliphatic carbon atoms. In yet other embodiments,the alkyl, alkenyl, and alkynyl groups employed in the invention contain1-8 aliphatic carbon atoms. In still other embodiments, the alkyl,alkenyl, and alkynyl groups employed in the invention contain 1-6aliphatic carbon atoms. In yet other embodiments, the alkyl, alkenyl,and alkynyl groups employed in the invention contain 1-4 carbon atoms.Illustrative aliphatic groups thus include, but are not limited to, forexample, methyl, ethyl, n-propyl, isopropyl, cyclopropyl,—CH₂-cyclopropyl, vinyl, allyl, n-butyl, sec-butyl, isobutyl,tert-butyl, cyclobutyl, —CH₂-cyclobutyl, n-pentyl, sec-pentyl,isopentyl, tert-pentyl, cyclopentyl, —CH₂-cyclopentyl, n-hexyl,sec-hexyl, cyclohexyl, —CH₂-cyclohexyl moieties and the like, whichagain, may bear one or more substituents. Alkenyl groups include, butare not limited to, for example, ethenyl, propenyl, butenyl,1-methyl-2-buten-1-yl, and the like. Representative alkynyl groupsinclude, but are not limited to, ethynyl, 2-propynyl (propargyl),1-propynyl, and the like.

Some examples of substituents of the above-described aliphatic (andother) moieties of compounds of the invention include, but are notlimited to aliphatic; heteroaliphatic; aryl; heteroaryl; arylalkyl;heteroarylalkyl; alkoxy; aryloxy; heteroalkoxy; heteroaryloxy;alkylthio; arylthio; heteroalkylthio; heteroarylthio; —F; —Cl; —Br, —I;—OH; —NO₂; —CN; —CF₃; —CH₂CF₃; —CHCl₂; —CH₂OH; —CH₂CH₂OH; —CH₂NH₂;—CH₂SO₂CH₃; —C(O)R_(x); —CO₂(R_(x)); —CON(R_(x))₂; —OC(O)R_(x);—OCO₂R_(x); —OCON(R_(x))₂; —N(R_(x))₂; —S(O)₂R_(x); —NR_(x)(CO)R_(x)wherein each occurrence of R_(x) independently includes, but is notlimited to, aliphatic, heteroaliphatic, aryl, heteroaryl, arylalkyl, orheteroarylalkyl, wherein any of the aliphatic, heteroaliphatic,arylalkyl, or heteroarylalkyl substituents described above and hereinmay be substituted or unsubstituted, branched or unbranched, cyclic oracyclic, and wherein any of the aryl or heteroaryl substituentsdescribed above and herein may be substituted or unsubstituted.Additional examples of generally applicable substituents are illustratedby the specific embodiments described herein.

The term “heteroaliphatic,” as used herein, refers to aliphatic moietiesthat contain one or more oxygen, sulfur, nitrogen, phosphorus, orsilicon atoms, e.g., in place of carbon atoms. Heteroaliphatic moietiesmay be branched, unbranched, cyclic or acyclic and include saturated andunsaturated heterocycles such as morpholino, pyrrolidinyl, etc. Incertain embodiments, heteroaliphatic moieties are substituted byindependent replacement of one or more of the hydrogen atoms thereonwith one or more moieties including, but not limited to aliphatic;heteroaliphatic; aryl; heteroaryl; arylalkyl; heteroarylalkyl; alkoxy;aryloxy; heteroalkoxy; heteroaryloxy; alkylthio; arylthio;heteroalkylthio; heteroarylthio; —F; —Cl; —Br; —I; —OH; —NO₂; —CN; —CF₃;—CH₂CF₃; —CHCl₂; —CH₂OH; —CH₂CH₂OH; —CH₂NH₂; —CH₂SO₂CH₃; —C(O)R_(x);—CO₂(R_(x)); —CON(R_(x))₂; —OC(O)R₂; —OCO₂R_(x); —OCON(R_(x));—N(R_(x))₂; —S(O)₂R_(x); —NR_(x)(CO)R_(x), wherein each occurrence of R,independently includes, but is not limited to, aliphatic,heteroaliphatic, aryl, heteroaryl, arylalkyl, or heteroarylalkyl,wherein any of the aliphatic, heteroaliphatic, arylalkyl, orheteroarylalkyl substituents described above and herein may besubstituted or unsubstituted, branched or unbranched, cyclic or acyclic,and wherein any of the aryl or heteroaryl substituents described aboveand herein may be substituted or unsubstituted. Additional examples ofgenerally applicable substitutents are illustrated by the specificembodiments described herein.

The terms “halo” and “halogen” as used herein refer to an atom selectedfrom fluorine, chlorine, bromine, and iodine.

The term “alkyl” includes saturated aliphatic groups, includingstraight-chain alkyl groups (e.g., methyl, ethyl, propyl, butyl, pentyl,hexyl, heptyl, octyl, nonyl, decyl, etc.), branched-chain alkyl groups(isopropyl, tert-butyl, isobutyl, etc.), cycloalkyl (alicyclic) groups(cyclopropyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl), alkylsubstituted cycloalkyl groups, and cycloalkyl substituted alkyl groups.In certain embodiments, a straight chain or branched chain alkyl has 6or fewer carbon atoms in its backbone (e.g., C₁-C₆ for straight chain,C₃-C₆ for branched chain), and more preferably 4 or fewer. Likewise,preferred cycloalkyls have from 3-8 carbon atoms in their ringstructure, and more preferably have 5 or 6 carbons in the ringstructure. The term C₁-C₆ includes alkyl groups containing 1 to 6 carbonatoms.

Moreover, unless otherwise specified, the term alkyl includes both“unsubstituted alkyls” and “substituted alkyls,” the latter of whichrefers to alkyl moieties having independently selected substituentsreplacing a hydrogen on one or more carbons of the hydrocarbon backbone.Such substituents can include, for example, alkenyl, alkynyl, halogen,hydroxyl, alkylcarbonyloxy, arylcarbonyloxy, alkoxycarbonyloxy,aryloxycarbonyloxy, carboxylate, alkylcarbonyl, arylcarbonyl,alkoxycarbonyl, aminocarbonyl, alkylaminocarbonyl, dialkylaminocarbonyl,alkylthiocarbonyl, alkoxyl, phosphate, phosphonato, phosphinato, cyano,amino (including alkyl amino, dialkylamino, arylamino, diarylamino, andalkylarylamino), acylamino (including alkylcarbonylamino,arylcarbonylamino, carbamoyl and ureido), amidino, imino, sulfhydryl,alkylthio, arylthio, thiocarboxylate, sulfates, alkylsulfinyl,sulfonato, sulfamoyl, sulfonamido, nitro, trifluoromethyl, cyano, azido,heterocyclyl, alkylaryl, or an aromatic or heteroaromatic moiety.Cycloalkyls can be further substituted, e.g., with the substituentsdescribed above. An “alkylaryl” or an “arylalkyl” moiety is an alkylsubstituted with an aryl (e.g., phenylmethyl (benzyl)). The term “alkyl”also includes the side chains of natural and unnatural amino acids. Theterm “n-alkyl” means a straight chain (i.e., unbranched) unsubstitutedalkyl group.

The term “alkenyl” includes unsaturated aliphatic groups analogous inlength and possible substitution to the alkyls described above, but thatcontain at least one double bond. For example, the term “alkenyl”includes straight-chain alkenyl groups (e.g., ethylenyl, propenyl,butenyl, pentenyl, hexenyl, heptenyl, octenyl, nonenyl, decenyl, etc.),branched-chain alkenyl groups, cycloalkenyl (alicyclic) groups(cyclopropenyl, cyclopentenyl, cyclohexenyl, cycloheptenyl,cyclooctenyl), alkyl or alkenyl substituted cycloalkenyl groups, andcycloalkyl or cycloalkenyl substituted alkenyl groups. In certainembodiments, a straight chain or branched chain alkenyl group has 6 orfewer carbon atoms in its backbone (e.g., C₂-C₆ for straight chain,C₃-C₆ for branched chain). Likewise, cycloalkenyl groups may have from3-8 carbon atoms in their ring structure, and more preferably have 5 or6 carbons in the ring structure. The term C₂-C₆ includes alkenyl groupscontaining 2 to 6 carbon atoms.

Moreover, unless otherwise specified, the term alkenyl includes both“unsubstituted alkenyls” and “substituted alkenyls,” the latter of whichrefers to alkenyl moieties having independently selected substituentsreplacing a hydrogen on one or more carbons of the hydrocarbon backbone.Such substituents can include, for example, alkyl groups, alkynylgroups, halogens, hydroxyl, alkylcarbonyloxy, arylcarbonyloxy,alkoxycarbonyloxy, aryloxycarbonyloxy, carboxylate, alkylcarbonyl,arylcarbonyl, alkoxycarbonyl, aminocarbonyl, alkylaminocarbonyl,dialkylaminocarbonyl, alkylthiocarbonyl, alkoxyl, phosphate,phosphonato, phosphinato, cyano, amino (including alkyl amino,dialkylamino, arylamino, diarylamino, and alkylarylamino), acylamino(including alkylcarbonylamino, arylcarbonylamino, carbamoyl and ureido),amidino, imino, sulfhydryl, alkylthio, arylthio, thiocarboxylate,sulfates, alkylsulfinyl, sulfonato, sulfamoyl, sulfonamido, nitro,trifluoromethyl, cyano, azido, heterocyclyl, alkylaryl, or an aromaticor heteroaromatic moiety.

The term “alkynyl” includes unsaturated aliphatic groups analogous inlength and possible substitution to the alkyls described above, butwhich contain at least one triple bond. For example, the term “alkynyl”includes straight-chain alkynyl groups (e.g., ethynyl, propynyl,butynyl, pentynyl, hexynyl, heptynyl, octynyl, nonynyl, decynyl, etc.),branched-chain alkynyl groups, and cycloalkyl or cycloalkenylsubstituted alkynyl groups. In certain embodiments, a straight chain orbranched chain alkynyl group has 6 or fewer carbon atoms in its backbone(e.g., C₂-C₆ for straight chain, C₃-C₆ for branched chain). The termC₂-C₆ includes alkynyl groups containing 2 to 6 carbon atoms.

Moreover, unless otherwise specified, the term alkynyl includes both“unsubstituted alkynyls” and “substituted alkynyls,” the latter of whichrefers to alkynyl moieties having independently selected substituentsreplacing a hydrogen on one or more carbons of the hydrocarbon backbone.Such substituents can include, for example, alkyl groups, alkynylgroups, halogens, hydroxyl, alkylcarbonyloxy, arylcarbonyloxy,alkoxycarbonyloxy, aryloxycarbonyloxy, carboxylate, alkylcarbonyl,arylcarbonyl, alkoxycarbonyl, aminocarbonyl, alkylaminocarbonyl,dialkylaminocarbonyl, alkylthiocarbonyl, alkoxyl, phosphate,phosphonato, phosphinato, cyano, amino (including alkyl amino,dialkylamino, arylamino, diarylamino, and alkylarylamino), acylamino(including alkylcarbonylamino, arylcarbonylamino, carbamoyl and ureido),amidino, imino, sulfhydryl, alkylthio, arylthio, thiocarboxylate,sulfates, alkylsulfinyl, sulfonato, sulfamoyl, sulfonamido, nitro,trifluoromethyl, cyano, azido, heterocyclyl, alkylaryl, or an aromaticor heteroaromatic moiety.

Unless the number of carbons is otherwise specified, “lower alkyl” asused herein means an alkyl group, as defined above, but having from oneto five carbon atoms in its backbone structure. “Lower alkenyl” and“lower alkynyl” have chain lengths of, for example, 2-5 carbon atoms.

The term “alkoxy” includes substituted and unsubstituted alkyl, alkenyl,and alkynyl groups covalently linked to an oxygen atom. Examples ofalkoxy groups include methoxy, ethoxy, isopropyloxy, propoxy, butoxy,and pentoxy groups. Examples of substituted alkoxy groups includehalogenated alkoxy groups. The alkoxy groups can be substituted withindependently selected groups such as alkenyl, alkynyl, halogen,hydroxyl, alkylcarbonyloxy, arylcarbonyloxy, alkoxycarbonyloxy,aryloxycarbonyloxy, carboxylate, alkylcarbonyl, arylcarbonyl,alkoxycarbonyl, aminocarbonyl, alkylaminocarbonyl, dialkylaminocarbonyl,alkylthiocarbonyl, alkoxyl, phosphate, phosphonato, phosphinato, cyano,amino (including alkyl amino, dialkylamino, arylamino, diarylamino, andalkylarylamino), acylamino (including alkylcarbonylamino,arylcarbonylamino, carbamoyl and ureido), amidino, imino, sulfflydryl,alkylthio, arylthio, thiocarboxylate, sulfates, alkylsulfinyl,sulfonato, sulfamoyl, sulfonamido, nitro, trifluoromethyl, cyano, azido,heterocyclyl, alkylaryl, or an aromatic or heteroaromatic moieties.Examples of halogen substituted alkoxy groups include, but are notlimited to, fluoromethoxy, difluoromethoxy, trifluoromethoxy,chloromethoxy, dichloromethoxy, trichloromethoxy, etc.

The term “heteroatom” includes atoms of any element other than carbon orhydrogen. Preferred heteroatoms are nitrogen, oxygen, sulfur andphosphorus.

The term “hydroxy” or “hydroxyl” includes groups with an —OH or —O—(with an appropriate counterion).

The term “halogen” includes fluorine, bromine, chlorine, iodine, etc.The term “perhalogenated” generally refers to a moiety wherein allhydrogens are replaced by halogen atoms.

The term “substituted” includes independently selected substituentswhich can be placed on the moiety and which allow the molecule toperform its intended function.

Examples of substituents include alkyl, alkenyl, alkynyl, aryl,(CR′R″)₀₋₃NR′R″, (CR′R″)₀₋₃ CN, NO₂, halogen, (CR′R″)₀₋₃C(halogen)₃,(CR′R″)₀₋₃CH(halogen)₂, (CR′R″)₀₋₃CH₂(halogen), (CR′R″)₀₋₃CONR′R″,(CR′R″)₀₋₃S(O)₁₋₂NR′R″, (CR′R″)₀₋₃CHO, (CR′R″)₀₋₃O(CR′R″)₀₋₃H,(CR′R″)₀₋₃S(O)₀₋₂R′, (CR′R″)₀₋₃O(CR′R″)₀₋₃H, (CR′R″)₀₋₃COR′,(CR′R″)₀₋₃CO₂R′, or (CR′R″)₀₋₃OR′ groups; wherein each R′ and R″ areeach independently hydrogen, a C₁-C₅ alkyl, C₂-C₅ alkenyl, C₂-C₅alkynyl, or aryl group, or R′ and R″ taken together are a benzylidenegroup or a —(CH₂)₂O(CH₂)₂— group.

The term “amine” or “amino” includes compounds or moieties in which anitrogen atom is covalently bonded to at least one carbon or heteroatom.The term “alkyl amino” includes groups and compounds wherein thenitrogen is bound to at least one additional alkyl group. The term“dialkyl amino” includes groups wherein the nitrogen atom is bound to atleast two additional alkyl groups.

The term “ether” includes compounds or moieties which contain an oxygenbonded to two different carbon atoms or heteroatoms. For example, theterm includes “alkoxyalkyl,” which refers to an alkyl, alkenyl, oralkynyl group covalently bonded to an oxygen atom which is covalentlybonded to another alkyl group.

The terms “polynucleotide,” “nucleotide sequence,” “nucleic acid,”“nucleic acid molecule,” “nucleic acid sequence,” and “oligonucleotide”refer to a polymer of two or more nucleotides. The polynucleotides canbe DNA, RNA, or derivatives or modified versions thereof. Thepolynucleotide may be single-stranded or double-stranded. Thepolynucleotide can be modified at the base moiety, sugar moiety, orphosphate backbone, for example, to improve stability of the molecule,its hybridization parameters, etc. The polynucleotide may comprise amodified base moiety which is selected from the group including but notlimited to 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil,hypoxanthine, xanthine, 4-acetylcytosine, 5-(carboxyhydroxylmethyl)uracil, 5-carboxymethylaminomethyl-2-thiouridine,5-carboxymethylaminomethyluracil, dihydrouracil,beta-D-galactosylqueosine, inosine, N6-isopentenyladenine,1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine,2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine,7-methylguanine, 5-methylaminomethyluracil,5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine,5′-methoxycarboxymethyluracil, 5-methoxyuracil,2-methylthio-N6-isopentenyladenine, wybutoxosine, pseudouracil,queosine, 2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil,4-thiouracil, 5-methyluracil, uracil-5-oxyacetic acid methylester,uracil-5-oxyacetic acid, 5-methyl-2-thiouracil,3-(3-amino-3-N-2-carboxypropyl) uracil, and 2,6-diaminopurine. Theolynucleotide may comprise a modified sugar moiety (e.g.,2′-fluororibose, ribose, 2′-deoxyribose, 2′-O-methylcytidine, arabinose,and hexose), and/or a modified phosphate moiety (e.g., phosphorothioatesand 5′-N-phosphoramidite linkages). A nucleotide sequence typicallycarries genetic information, including the information used by cellularmachinery to make proteins and enzymes. These terms include double- orsingle-stranded genomic and cDNA, RNA, any synthetic and geneticallymanipulated polynucleotide, and both sense and antisensepolynucleotides. This includes single- and double-stranded molecules,i.e., DNA-DNA, DNA-RNA, and RNA-RNA hybrids, as well as “protein nucleicacids” (PNA) formed by conjugating bases to an amino acid backbone.

The term “base” includes the known purine and pyrimidine heterocyclicbases, deazapurines, and analogs (including heterocyclic substitutedanalogs, e.g., aminoethyoxy phenoxazine), derivatives (e.g., 1-alkyl-,1-alkenyl-, heteroaromatic- and 1-alkynyl derivatives) and tautomersthereof. Examples of purines include adenine, guanine, inosine,diaminopurine, and xanthine and analogs (e.g., 8-oxo-N⁶-methyladenine or7-diazaxanthine) and derivatives thereof. Pyrimidines include, forexample, thymine, uracil, and cytosine, and their analogs (e.g.,5-methylcytosine, 5-methyluracil, 5-(l-propynyl)uracil,5-(l-propynyl)cytosine and 4,4-ethanocytosine). Other examples ofsuitable bases include non-purinyl and non-pyrimidinyl bases such as2-aminopyridine and triazines.

In a preferred embodiment, the nucleomonomers of an oligonucleotide ofthe invention are RNA nucleotides. In another preferred embodiment, thenucleomonomers of an oligonucleotide of the invention are modified RNAnucleotides. Thus, the oligonucleotides contain modified RNAnucleotides.

The term “nucleoside” includes bases which are covalently attached to asugar moiety, preferably ribose or deoxyribose. Examples of preferrednucleosides include ribonucleosides and deoxyribonucleosides.Nucleosides also include bases linked to amino acids or amino acidanalogs which may comprise free carboxyl groups, free amino groups, orprotecting groups. Suitable protecting groups are well known in the art(see P. G. M. Wuts and T. W. Greene, “Protective Groups in OrganicSynthesis”, 2^(nd) Ed., Wiley-Interscience, New York, 1999).

The term “nucleotide” includes nucleosides which further comprise aphosphate group or a phosphate analog.

The nucleic acid molecules may be associated with a hydrophobic moietyfor targeting and/or delivery of the molecule to a cell. In certainembodiments, the hydrophobic moiety is associated with the nucleic acidmolecule through a linker. In certain embodiments, the association isthrough non-covalent interactions. In other embodiments, the associationis through a covalent bond. Any linker known in the art may be used toassociate the nucleic acid with the hydrophobic moiety. Linkers known inthe art are described in published international PCT applications, WO92/03464, WO 95/23162, WO 2008/021157, WO 2009/021157, WO 2009/134487,WO 2009/126933, U.S. Patent Application Publication 2005/0107325, U.S.Pat. No. 5,414,077, U.S. Pat. No. 5,419,966, U.S. Pat. No. 5,512,667,U.S. Pat. No. 5,646,126, and U.S. Pat. No. 5,652,359, which areincorporated herein by reference. The linker may be as simple as acovalent bond to a multi-atom linker. The linker may be cyclic oracyclic. The linker may be optionally substituted. In certainembodiments, the linker is capable of being cleaved from the nucleicacid. In certain embodiments, the linker is capable of being hydrolyzedunder physiological conditions. In certain embodiments, the linker iscapable of being cleaved by an enzyme (e.g., an esterase orphosphodiesterase). In certain embodiments, the linker comprises aspacer element to separate the nucleic acid from the hydrophobic moiety.The spacer element may include one to thirty carbon or heteroatoms. Incertain embodiments, the linker and/or spacer element comprisesprotonatable functional groups. Such protonatable functional groups maypromote the endosomal escape of the nucleic acid molecule. Theprotonatable functional groups may also aid in the delivery of thenucleic acid to a cell, for example, neutralizing the overall charge ofthe molecule. In other embodiments, the linker and/or spacer element isbiologically inert (that is, it does not impart biological activity orfunction to the resulting nucleic acid molecule).

In certain embodiments, the nucleic acid molecule with a linker andhydrophobic moiety is of the formulae described herein. In certainembodiments, the nucleic acid molecule is of the formula:

wherein

X is N or CH;

A is a bond; substituted or unsubstituted, cyclic or acyclic, branchedor unbranched aliphatic; or substituted or unsubstituted, cyclic oracyclic, branched or unbranched heteroaliphatic;

R¹ is a hydrophobic moiety;

-   -   R² is hydrogen; an oxygen-protecting group; cyclic or acyclic,        substituted or unsubstituted, branched or unbranched aliphatic;        cyclic or acyclic, substituted or unsubstituted, branched or        unbranched heteroaliphatic; substituted or unsubstituted,        branched or unbranched acyl; substituted or unsubstituted,        branched or unbranched aryl; substituted or unsubstituted,        branched or unbranched heteroaryl; and    -   R³ is a nucleic acid.    -   In certain embodiments, the molecule is of the formula:

-   -   In certain embodiments, the molecule is of the formula:

-   -   In certain embodiments, the molecule is of the formula:

-   -   In certain embodiments, the molecule is of the formula:

-   -   In certain embodiments, X is N. In certain embodiments, X is CH.    -   In certain embodiments, A is a bond. In certain embodiments, A        is substituted or unsubstituted, cyclic or acyclic, branched or        unbranched aliphatic. In certain embodiments, A is acyclic,        substituted or unsubstituted, branched or unbranched aliphatic.        In certain embodiments, A is acyclic, substituted, branched or        unbranched aliphatic. In certain embodiments, A is acyclic,        substituted, unbranched aliphatic. In certain embodiments, A is        acyclic, substituted, unbranched alkyl. In certain embodiments,        A is acyclic, substituted, unbranched C₁₋₂₀ alkyl. In certain        embodiments, A is acyclic, substituted, unbranched C₁₋₁₂ alkyl.        In certain embodiments, A is acyclic, substituted, unbranched        C₁₋₁₀ alkyl. In certain embodiments, A is acyclic, substituted,        unbranched C₁₋₈ alkyl. In certain embodiments, A is acyclic,        substituted, unbranched C₁₋₆ alkyl. In certain embodiments, A is        substituted or unsubstituted, cyclic or acyclic, branched or        unbranched heteroaliphatic. In certain embodiments, A is        acyclic, substituted or unsubstituted, branched or unbranched        heteroaliphatic. In certain embodiments, A is acyclic,        substituted, branched or unbranched heteroaliphatic. In certain        embodiments, A is acyclic, substituted, unbranched        heteroaliphatic.    -   In certain embodiments, A is of the formula:

-   -   In certain embodiments, A is of one of the formulae:

-   -   In certain embodiments, A is of one of the formulae:

-   -   In certain embodiments, A is of one of the formulae:

-   -   In certain embodiments, A is of the formula:

-   -   In certain embodiments, A is of the formula:

-   -   In certain embodiments, A is of the formula:

wherein

-   -   each occurrence of R is independently the side chain of a        natural or unnatural amino acid; and    -   n is an integer between 1 and 20, inclusive. In certain        embodiments, A is of the formula:

In certain embodiments, each occurrence of R is independently the sidechain of a natural amino acid. In certain embodiments, n is an integerbetween 1 and 15, inclusive. In certain embodiments, n is an integerbetween 1 and 10, inclusive. In certain embodiments, n is an integerbetween 1 and 5, inclusive.

-   -   In certain embodiments, A is of the formula:

wherein n is an integer between 1 and 20, inclusive. In certainembodiments, A is of the formula:

In certain embodiments, n is an integer between 1 and 15, inclusive. Incertain embodiments, n is an integer between 1 and 10, inclusive. Incertain embodiments, n is an integer between 1 and 5, inclusive.

-   -   In certain embodiments, A is of the formula:

wherein n is an integer between 1 and 20, inclusive. In certainembodiments, A is of the formula:

In certain embodiments, n is an integer between 1 and 15, inclusive. Incertain embodiments, n is an integer between 1 and 10, inclusive. Incertain embodiments, n is an integer between 1 and 5, inclusive.

-   -   In certain embodiments, the molecule is of the formula:

wherein X, R¹, R², and R³ are as defined herein; and

-   -   A′ is substituted or unsubstituted, cyclic or acyclic, branched        or unbranched aliphatic; or substituted or unsubstituted, cyclic        or acyclic, branched or unbranched heteroaliphatic.    -   In certain embodiments, A′ is of one of the formulae:

In certain embodiments, A is of one of the formulae:

In certain embodiments, A is of one of the formulae:

In certain embodiments, A is of the formula:

In certain embodiments, A is of the formula:

In certain embodiments, R¹ is a steroid. In certain embodiments, R¹ is acholesterol. In certain embodiments, R¹ is a lipophilic vitamin. Incertain embodiments, R¹ is a vitamin A. In certain embodiments, R¹ is avitamin E.

In certain embodiments, R¹ is of the formula:

wherein R^(A) is substituted or unsubstituted, cyclic or acyclic,branched or unbranched aliphatic; or substituted or unsubstituted,cyclic or acyclic, branched or unbranched heteroaliphatic.

In certain embodiments, R¹ is of the formula:

In certain embodiments, R¹ is of the formula:

In certain embodiments, R¹ is of the formula:

In certain embodiments, R¹ is of the formula:

In certain embodiments, R¹ is of the formula:

In certain embodiments, the nucleic acid molecule is of the formula:

wherein

X is N or CH;

A is a bond; substituted or unsubstituted, cyclic or acyclic, branchedor unbranched aliphatic; or substituted or unsubstituted, cyclic oracyclic, branched or unbranched heteroaliphatic;

R¹ is a hydrophobic moiety;

R² is hydrogen; an oxygen-protecting group; cyclic or acyclic,substituted or unsubstituted, branched or unbranched aliphatic; cyclicor acyclic, substituted or unsubstituted, branched or unbranchedheteroaliphatic; substituted or unsubstituted, branched or unbranchedacyl; substituted or unsubstituted, branched or unbranched aryl;substituted or unsubstituted, branched or unbranched heteroaryl; and

R³ is a nucleic acid.

In certain embodiments, the nucleic acid molecule is of the formula:

wherein

X is N or CH;

A is a bond; substituted or unsubstituted, cyclic or acyclic, branchedor unbranched aliphatic; or substituted or unsubstituted, cyclic oracyclic, branched or unbranched heteroaliphatic;

R¹ is a hydrophobic moiety;

R² is hydrogen; an oxygen-protecting group; cyclic or acyclic,substituted or unsubstituted, branched or unbranched aliphatic; cyclicor acyclic, substituted or unsubstituted, branched or unbranchedheteroaliphatic; substituted or unsubstituted, branched or unbranchedacyl; substituted or unsubstituted, branched or unbranched aryl;substituted or unsubstituted, branched or unbranched heteroaryl; and

R³ is a nucleic acid.

In certain embodiments, the nucleic acid molecule is of the formula:

wherein

X is N or CH;

A is a bond; substituted or unsubstituted, cyclic or acyclic, branchedor unbranched aliphatic; or substituted or unsubstituted, cyclic oracyclic, branched or unbranched heteroaliphatic;

R¹ is a hydrophobic moiety;

R² is hydrogen; an oxygen-protecting group; cyclic or acyclic,substituted or unsubstituted, branched or unbranched aliphatic; cyclicor acyclic, substituted or unsubstituted, branched or unbranchedheteroaliphatic; substituted or unsubstituted, branched or unbranchedacyl; substituted or unsubstituted, branched or unbranched aryl;substituted or unsubstituted, branched or unbranched heteroaryl; and

R³ is a nucleic acid. In certain embodiments, the nucleic acid moleculeis of the formula:

In certain embodiments, the nucleic acid molecule is of the formula:

In certain embodiments, the nucleic acid molecule is of the formula:

wherein R³ is a nucleic acid.

In certain embodiments, the nucleic acid molecule is of the formula:

wherein R³ is a nucleic acid; and

n is an integer between 1 and 20, inclusive.

In certain embodiments, the nucleic acid molecule is of the formula:

In certain embodiments, the nucleic acid molecule is of the formula:

In certain embodiments, the nucleic acid molecule is of the formula:

In certain embodiments, the nucleic acid molecule is of the formula:

In certain embodiments, the nucleic acid molecule is of the formula:

As used herein, the term “linkage” includes a naturally occurring,unmodified phosphodiester moiety (—O—(PO²—)—O—) that covalently couplesadjacent nucleomonomers. As used herein, the term “substitute linkage”includes any analog or derivative of the native phosphodiester groupthat covalently couples adjacent nucleomonomers. Substitute linkagesinclude phosphodiester analogs, e.g., phosphorothioate,phosphorodithioate, and P-ethyoxyphosphodiester, P-ethoxyphosphodiester,P-alkyloxyphosphotriester, methylphosphonate, and nonphosphoruscontaining linkages, e.g., acetals and amides. Such substitute linkagesare known in the art (e.g., Bjergarde et al. 1991. Nucleic Acids Res.19:5843; Caruthers et al. 1991. Nucleosides Nucleotides. 10:47). Incertain embodiments, non-hydrolizable linkages are preferred, such asphosphorothiate linkages.

In certain embodiments, oligonucleotides of the invention comprisehydrophobically modified nucleotides or “hydrophobic modifications.” Asused herein “hydrophobic modifications” refers to bases that aremodified such that (1) overall hydrophobicity of the base issignificantly increased, and/or (2) the base is still capable of formingclose to regular Watson-Crick interaction. Several non-limiting examplesof base modifications include 5-position uridine and cytidinemodifications such as phenyl, 4-pyridyl, 2-pyridyl, indolyl, andisobutyl, phenyl (C6H5OH); tryptophanyl (C8H6N)CH2CH(NH2)CO), Isobutyl,butyl, aminobenzyl; phenyl; and naphthyl.

Another type of conjugates that can be attached to the end (3′ or 5′end), the loop region, or any other parts of the sd-rxRNA might includea sterol, sterol type molecule, peptide, small molecule, protein, etc.In some embodiments, a sd-rxRNA may contain more than one conjugates(same or different chemical nature). In some embodiments, the conjugateis cholesterol.

Another way to increase target gene specificity, or to reduce off-targetsilencing effect, is to introduce a 2′-modification (such as the 2′-Omethyl modification) at a position corresponding to the second 5′-endnucleotide of the guide sequence. Antisense (guide) sequences of theinvention can be “chimeric oligonucleotides” which comprise an RNA-likeand a DNA-like region.

The language “RNase H activating region” includes a region of anoligonucleotide, e.g., a chimeric oligonucleotide, that is capable ofrecruiting RNase H to cleave the target RNA strand to which theoligonucleotide binds. Typically, the RNase activating region contains aminimal core (of at least about 3-5, typically between about 3-12, moretypically, between about 5-12, and more preferably between about 5-10contiguous nucleomonomers) of DNA or DNA-like nucleomonomers. (See,e.g., U.S. Pat. No. 5,849,902). Preferably, the RNase H activatingregion comprises about nine contiguous deoxyribose containingnucleomonomers.

The language “non-activating region” includes a region of an antisensesequence, e.g., a chimeric oligonucleotide, that does not recruit oractivate RNase H. Preferably, a non-activating region does not comprisephosphorothioate DNA. The oligonucleotides of the invention comprise atleast one non-activating region. In one embodiment, the non-activatingregion can be stabilized against nucleases or can provide specificityfor the target by being complementary to the target and forming hydrogenbonds with the target nucleic acid molecule, which is to be bound by theoligonucleotide.

In one embodiment, at least a portion of the contiguous polynucleotidesare linked by a substitute linkage, e.g., a phosphorothioate linkage.

In certain embodiments, most or all of the nucleotides beyond the guidesequence (2′-modified or not) are linked by phosphorothioate linkages.Such constructs tend to have improved pharmacokinetics due to theirhigher affinity for serum proteins. The phosphorothioate linkages in thenon-guide sequence portion of the polynucleotide generally do notinterfere with guide strand activity, once the latter is loaded intoRISC. It is surprisingly demonstrated herein that high levels ofphosphorothioate modification can lead to improved delivery. In someembodiments, the guide and/or passenger strand is completelyphosphorothioated.

Antisense (guide) sequences of the present invention may include“morpholino oligonucleotides.” Morpholino oligonucleotides are non-ionicand function by an RNase H-independent mechanism. Each of the 4 geneticbases (Adenine, Cytosine, Guanine, and Thymine/Uracil) of the morpholinooligonucleotides is linked to a 6-membered morpholine ring. Morpholinooligonucleotides are made by joining the 4 different subunit types by,e.g., non-ionic phosphorodiamidate inter-subunit linkages. Morpholinooligonucleotides have many advantages including: complete resistance tonucleases (Antisense & Nucl. Acid Drug Dev. 1996. 6:267); predictabletargeting (Biochemica Biophysica Acta. 1999. 1489:141); reliableactivity in cells (Antisense & Nucl. Acid Drug Dev. 1997. 7:63);excellent sequence specificity (Antisense & Nucl. Acid Drug Dev. 1997.7:151); minimal non-antisense activity (Biochemica Biophysica Acta.1999. 1489:141); and simple osmotic or scrape delivery (Antisense &Nucl. Acid Drug Dev. 1997. 7:291). Morpholino oligonucleotides are alsopreferred because of their non-toxicity at high doses. A discussion ofthe preparation of morpholino oligonucleotides can be found in Antisense& Nucl. Acid Drug Dev. 1997. 7:187.

The chemical modifications described herein are believed, based on thedata described herein, to promote single stranded polynucleotide loadinginto the RISC. Single stranded polynucleotides have been shown to beactive in loading into RISC and inducing gene silencing. However, thelevel of activity for single stranded polynucleotides appears to be 2 to4 orders of magnitude lower when compared to a duplex polynucleotide.

The present invention provides a description of the chemicalmodification patterns, which may (a) significantly increase stability ofthe single stranded polynucleotide (b) promote efficient loading of thepolynucleotide into the RISC complex and (c) improve uptake of thesingle stranded nucleotide by the cell. The chemical modificationpatterns may include combination of ribose, backbone, hydrophobicnucleoside and conjugate type of modifications. In addition, in some ofthe embodiments, the 5′ end of the single polynucleotide may bechemically phosphorylated.

In yet another embodiment, the present invention provides a descriptionof the chemical modifications patterns, which improve functionality ofRISC inhibiting polynucleotides. Single stranded polynucleotides havebeen shown to inhibit activity of a preloaded RISC complex through thesubstrate competition mechanism. For these types of molecules,conventionally called antagomers, the activity usually requires highconcentration and in vivo delivery is not very effective. The presentinvention provides a description of the chemical modification patterns,which may (a) significantly increase stability of the single strandedpolynucleotide (b) promote efficient recognition of the polynucleotideby the RISC as a substrate and/or (c) improve uptake of the singlestranded nucleotide by the cell. The chemical modification patterns mayinclude combination of ribose, backbone, hydrophobic nucleoside andconjugate type of modifications.

The modifications provided by the present invention are applicable toall polynucleotides. This includes single stranded RISC enteringpolynucleotides, single stranded RISC inhibiting polynucleotides,conventional duplexed polynucleotides of variable length (15-40 bp),asymmetric duplexed polynucleotides, and the like. Polynucleotides maybe modified with wide variety of chemical modification patterns,including 5′ end, ribose, backbone and hydrophobic nucleosidemodifications.

Aspects of the invention relate to nucleic acid molecules that arehighly modified with phosphorothioate backbone modifications. Completelyphosphorothioated compounds (21552) that are highly active weredisclosed in PCT Publication No. WO2011/119852, incorporated byreference herein. Interestingly, compounds that contained a 21 mer guidestrand that was completely phosphorothioate modified were active,however, reducing the guide strand length by two nucleotides, e.g., 19mer guide strand (21550), resulted in reduced activity. Without wishingto be bound by any theory, increasing the guide strand from 19 to 21nucleotides (fully phosphorothioated guide strands) may increase themelting temperature between the guide strand and mRNA, resulting inenhanced silencing activity. Varying phosphorothioate content on the 13mer passenger strand such that it was either completely phosphorothioatemodified or contained 6 phosphorothioate modifications did not result inaltered activity (21551 vs 21556 as disclosed in PCT Publication No.WO2011/119852).

Several past groups have tried to develop completely phosphorothioatedRNAi compounds. Completely phosphorothioated duplexes, lacking singlestranded regions, have been designed and tested before, however, thesecompounds did not demonstrate acceptable pharmacokinetic profiles.Completely phosphorothioated single stranded RNAi compounds have alsobeen designed previously, however, these compounds did not efficientlyenter RISC. PCT Publication No. WO2011/119852, incorporated by referenceherein in its entirety, disclosed hybrid RNAi compounds that efficientlyenter RISC and contain complete phosphorothioated backbones.

In some aspects, the disclosure relates to the discovery of isolateddouble stranded nucleic acid molecules having highly phosphorothioatedbackbones (e.g., having at least one strand with a completelyphosphorothioated backbone, or almost completely phosphorothioatedbackbone (e.g., having one un-phosphorothioated residue)). It wassurprisingly found herein that high levels of phosphorothioatemodifications mediate increased levels of cellular uptake of theisolated double stranded nucleic acid molecule in the central nervoussystem (CNS) relative to isolated double stranded nucleic acid moleculeshaving less phosphorothioate modifications (e.g. not having at least onestrand that is fully phosphorothioated or almost completelyphosphorothioated).

Synthesis

Oligonucleotides of the invention can be synthesized by any method knownin the art, e.g., using enzymatic synthesis and/or chemical synthesis.The oligonucleotides can be synthesized in vitro (e.g., using enzymaticsynthesis and chemical synthesis) or in viva (using recombinant DNAtechnology well known in the art).

In a preferred embodiment, chemical synthesis is used for modifiedpolynucleotides. Chemical synthesis of linear oligonucleotides is wellknown in the art and can be achieved by solution or solid phasetechniques. Preferably, synthesis is by solid phase methods.Oligonucleotides can be made by any of several different syntheticprocedures including the phosphoramidite, phosphite triester,H-phosphonate, and phosphotriester methods, typically by automatedsynthesis methods.

Oligonucleotide synthesis protocols are well known in the art and can befound, eg., in U.S. Pat. No. 5,830,653; WO 98/13526; Stec et al. 1984.J. Am. Chem. Soc. 106:6077; Stec et al. 1985. J. Org. Chem. 50:3908;Stec et al. J. Chromatog. 1985. 326:263; LaPlanche et al. 1986. Nucl.Acid. Res. 1986. 14:9081: Fasman G. D., 1989. Practical Handbook ofBiochemistry and Molecular Biology. 1989. CRC Press, Boca Raton, Fla.;Lamone. 1993. Biochem. Soc. Trans. 21:1; U.S. Pat. No. 5,013,830; U.S.Pat. No. 5,214,135; U.S. Pat. No. 5,525,719; Kawasaki et al. 1993. J.Med. Chem. 36:831; WO 92/03568; U.S. Pat. No. 5,276,019; and U.S. Pat.No. 5,264,423.

The synthesis method selected can depend on the length of the desiredoligonucleotide and such choice is within the skill of the ordinaryartisan. For example, the phosphoramidite and phosphite triester methodcan produce oligonucleotides having 175 or more nucleotides, while theH-phosphonate method works well for oligonucleotides of less than 100nucleotides. If modified bases are incorporated into theoligonucleotide, and particularly if modified phosphodiester linkagesare used, then the synthetic procedures are altered as needed accordingto known procedures. In this regard, Uhlmann et al. (1990, ChemicalReviews 90:543-584) provide references and outline procedures for makingoligonucleotides with modified bases and modified phosphodiesterlinkages. Other exemplary methods for making oligonucleotides are taughtin Sonveaux. 1994. “Protecting Groups in Oligonucleotide Synthesis”;Agrawal. Methods in Molecular Biology 26:1. Exemplary synthesis methodsare also taught in “Oligonucleotide Synthesis—A Practical Approach”(Gait, M. J. IRL Press at Oxford University Press. 1984). Moreover,linear oligonucleotides of defined sequence, including some sequenceswith modified nucleotides, are readily available from several commercialsources.

The oligonucleotides may be purified by polyacrylamide gelelectrophoresis, or by any of a number of chromatographic methods,including gel chromatography and high pressure liquid chromatography. Toconfirm a nucleotide sequence, especially unmodified nucleotidesequences, oligonucleotides may be subjected to DNA sequencing by any ofthe known procedures, including Maxam and Gilbert sequencing, Sangersequencing, capillary electrophoresis sequencing, the wandering spotsequencing procedure or by using selective chemical degradation ofoligonucleotides bound to Hybond paper. Sequences of shortoligonucleotides can also be analyzed by laser desorption massspectroscopy or by fast atom bombardment (McNeal, et al., 1982, J. Am.Chem. Soc. 104:976; Viari, et al., 1987, Biomed. Environ. Mass Spectrom.14:83; Grotjahn et al., 1982, Nuc. Acid Res. 10:4671). Sequencingmethods are also available for RNA oligonucleotides.

The quality of oligonucleotides synthesized can be verified by testingthe oligonucleotide by capillary electrophoresis and denaturing stronganion HPLC (SAX-HPLC) using, e.g., the method of Bergot and Egan. 1992.J. Chrom. 599:35.

Other exemplary synthesis techniques are well known in the art (see,e.g., Sambrook et al., Molecular Cloning: a Laboratory Manual, SecondEdition (1989); DNA Cloning, Volumes I and II (DN Glover Ed. 1985);Oligonucleotide Synthesis (M J Gait Ed, 1984; Nucleic Acid Hybridisation(B D Hames and S J Higgins eds. 1984); A Practical Guide to MolecularCloning (1984); or the series, Methods in Enzymology (Academic Press,Inc.)).

In certain embodiments, the subject RNAi constructs or at least portionsthereof are transcribed from expression vectors encoding the subjectconstructs. Any art recognized vectors may be use for this purpose. Thetranscribed RNAi constructs may be isolated and purified, before desiredmodifications (such as replacing an unmodified sense strand with amodified one, etc.) are carried out.

Uptake of Oligonucleotides by Cells

Oligonucleotides and oligonucleotide compositions are contacted with(i.e., brought into contact with, also referred to herein asadministered or delivered to) and taken up by one or more cells or acell lysate. The term “cells” includes prokaryotic and eukaryotic cells,preferably vertebrate cells, and, more preferably, mammalian cells. In apreferred embodiment, the oligonucleotide compositions of the inventionare contacted with human cells.

Oligonucleotide compositions of the invention can be contacted withcells in vitro, e.g., in a test tube or culture dish, (and may or maynot be introduced into a subject) or in vivo, e.g., in a subject such asa mammalian subject. In some embodiments, Oligonucleotides areadministered topically or through electroporation. Oligonucleotides aretaken up by cells at a slow rate by endocytosis, but endocytosedoligonucleotides are generally sequestered and not available, e.g., forhybridization to a target nucleic acid molecule. In one embodiment,cellular uptake can be facilitated by electroporation or calciumphosphate precipitation. However, these procedures are only useful forin vitro or ex vivo embodiments, are not convenient and, in some cases,are associated with cell toxicity.

In another embodiment, delivery of oligonucleotides into cells can beenhanced by suitable art recognized methods including calcium phosphate,DMSO, glycerol or dextran, electroporation, or by transfection, e.g.,using cationic, anionic, or neutral lipid compositions or liposomesusing methods known in the art (see e.g., WO 90/14074; WO 91/16024: WO91/17424, U.S. Pat. No. 4,897,355; Bergan et al. 1993. Nucleic AcidsResearch. 21:3567). Enhanced delivery of oligonucleotides can also bemediated by the use of vectors (See e.g., Shi, Y. 2003. Trends Genet2003 Jan. 19:9; Reichhart J M et al. Genesis. 2002. 34(1-2):1604, Yu elal. 2002. Proc. Natl. Acad Sci. USA 99:6047; Sui et al. 2002. Proc.Natl. Acad Sci. USA 99:5515) viruses, polyamine or polycation conjugatesusing compounds such as polylysine, protamine, or Ni, N12-bis (ethyl)spermine (see, e.g., Bartzatt, R. et al. 1989. Biotechnol. Appl.Biochem. 11:133; Wagner E. et al. 1992. Proc. Natl. Acad. Sci. 88:4255).

In certain embodiments, the sd-rxRNA of the invention may be deliveredby using various beta-glucan containing particles, referred to as GeRPs(glucan encapsulated RNA loaded particle), described in, andincorporated by reference from, U.S. Provisional Application No.61/310,611, filed on Mar. 4, 2010 and entitled “Formulations and Methodsfor Targeted Delivery to Phagocyte Cells.” Such particles are alsodescribed in, and incorporated by reference from US Patent PublicationsUS 2005/0281781 A1, and US 2010/0040656, U.S. Pat. No. 8,815,818 and inPCT publications WO 2006/007372, and WO 2007/050643. The sd-rxRNAmolecule may be hydrophobically modified and optionally may beassociated with a lipid and/or amphiphilic peptide. In certainembodiments, the beta-glucan particle is derived from yeast. In certainembodiments, the payload trapping molecule is a polymer, such as thosewith a molecular weight of at least about 1000 Da, 10,000 Da, 50,000 Da,100 kDa, 500 kDa, etc. Preferred polymers include (without limitation)cationic polymers, chitosans, or PEI (polyethylenimine), etc.

Glucan particles can be derived from insoluble components of fungal cellwalls such as yeast cell walls. In some embodiments, the yeast isBaker's yeast. Yeast-derived glucan molecules can include one or more ofß-(1,3)-Glucan, ß-(1,6)-Glucan, mannan and chitin. In some embodiments,a glucan particle comprises a hollow yeast cell wall whereby theparticle maintains a three dimensional structure resembling a cell,within which it can complex with or encapsulate a molecule such as anRNA molecule. Some of the advantages associated with the use of yeastcell wall particles are availability of the components, theirbiodegradable nature, and their ability to be targeted to phagocyticcells.

In some embodiments, glucan particles can be prepared by extraction ofinsoluble components from cell walls, for example by extracting Baker'syeast (Fleischmann's) with 1M NaOH/pH 4.0H2O, followed by washing anddrying. Methods of preparing yeast cell wall particles are discussed in,and incorporated by reference from U.S. Pat. Nos. 4,810,646, 4,992,540,5,082,936, 5,028,703, 5,032,401, 5,322,841, 5,401,727, 5,504,079,5,607,677, 5,968,811, 6,242,594, 6,444,448, 6,476,003, US PatentPublications 2003/0216346, 2004/0014715 and 2010/0040656, and PCTpublished application WO02/12348.

Protocols for preparing glucan particles are also described in, andincorporated by reference from, the following references: Soto andOstroff (2008), “Characterization of multilayered nanoparticlesencapsulated in yeast cell wall particles for DNA delivery.” BioconjugChem 19(4):840-8; Soto and Ostroff (2007), “Oral Macrophage MediatedGene Delivery System,” Nanotech, Volume 2, Chapter 5 (“Drug Delivery”),pages 378-381; and Li et al. (2007), “Yeast glucan particles activatemurine resident macrophages to secrete proinflammatory cytokines viaMyD88- and Syk kinase-dependent pathways.” Clinical Immunology 124(2):170-181.

Glucan containing particles such as yeast cell wall particles can alsobe obtained commercially. Several non-limiting examples include:Nutricell MOS 55 from Biorigin (Sao Paolo, Brazil), SAF-Mannan (SAFAgri, Minneapolis, Minn.), Nutrex (Sensient Technologies, Milwaukee,Wis.), alkali-extracted particles such as those produced by Nutricepts(Nutricepts Inc., Burnsville, Minn.) and ASA Biotech, acid-extracted WGPparticles from Biopolymer Engineering, and organic solvent-extractedparticles such as Adjuvax™ from Alpha-beta Technology, Inc. (Worcester,Mass.) and microparticulate glucan from Novogen (Stamford, Conn.).

Glucan particles such as yeast cell wall particles can have varyinglevels of purity depending on the method of production and/orextraction. In some instances, particles are alkali-extracted,acid-extracted or organic solvent-extracted to remove intracellularcomponents and/or the outer mannoprotein layer of the cell wall. Suchprotocols can produce particles that have a glucan (w/w) content in therange of 50%-90%. In some instances, a particle of lower purity, meaninglower glucan w/w content may be preferred, while in other embodiments, aparticle of higher purity, meaning higher glucan w/w content may bepreferred.

Glucan particles, such as yeast cell wall particles, can have a naturallipid content. For example, the particles can contain 1%, 2%, 3%, 4%,5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%,20% or more than 20% w/w lipid. In the Examples section, theeffectiveness of two glucan particle batches are tested: YGP SAF and YGPSAF+L (containing natural lipids). In some instances, the presence ofnatural lipids may assist in complexation or capture of RNA molecules.

Glucan containing particles typically have a diameter of approximately2-4 microns, although particles with a diameter of less than 2 micronsor greater than 4 microns are also compatible with aspects of theinvention.

The RNA molecule(s) to be delivered are complexed or “trapped” withinthe shell of the glucan particle. The shell or RNA component of theparticle can be labeled for visualization, as described in, andincorporated by reference from, Soto and Ostroff (2008) Bioconjug Chem19:840. Methods of loading GeRPs are discussed further below.

The optimal protocol for uptake of oligonucleotides will depend upon anumber of factors, the most crucial being the type of cells that arebeing used. Other factors that are important in uptake include, but arenot limited to, the nature and concentration of the oligonucleotide, theconfluence of the cells, the type of culture the cells are in (e.g., asuspension culture or plated) and the type of media in which the cellsare grown.

Encapsulating Agents

Encapsulating agents entrap oligonucleotides within vesicles. In anotherembodiment of the invention, an oligonucleotide may be associated with acarrier or vehicle, e.g., liposomes or micelles, although other carrierscould be used, as would be appreciated by one skilled in the art.Liposomes are vesicles made of a lipid bilayer having a structuresimilar to biological membranes. Such carriers are used to facilitatethe cellular uptake or targeting of the oligonucleotide, or improve theoligonucleotide's pharmacokinetic or toxicologic properties.

For example, the oligonucleotides of the present invention may also beadministered encapsulated in liposomes, pharmaceutical compositionswherein the active ingredient is contained either dispersed or variouslypresent in corpuscles consisting of aqueous concentric layers adherentto lipidic layers. The oligonucleotides, depending upon solubility, maybe present both in the aqueous layer and in the lipidic layer, or inwhat is generally termed a liposomic suspension. The hydrophobic layer,generally but not exclusively, comprises phopholipids such as lecithinand sphingomyelin, steroids such as cholesterol, more or less ionicsurfactants such as diacetylphosphate, stearylamine, or phosphatidicacid, or other materials of a hydrophobic nature. The diameters of theliposomes generally range from about 15 nm to about 5 microns.

The use of liposomes as drug delivery vehicles offers severaladvantages. Liposomes increase intracellular stability, increase uptakeefficiency and improve biological activity. Liposomes are hollowspherical vesicles composed of lipids arranged in a similar fashion asthose lipids which make up the cell membrane. They have an internalaqueous space for entrapping water soluble compounds and range in sizefrom 0.05 to several microns in diameter. Several studies have shownthat liposomes can deliver nucleic acids to cells and that the nucleicacids remain biologically active. For example, a lipid delivery vehicleoriginally designed as a research tool, such as Lipofectin orLIPOFECTAMINE™ 2000, can deliver intact nucleic acid molecules to cells.

Specific advantages of using liposomes include the following: they arenon-toxic and biodegradable in composition; they display longcirculation half-lives; and recognition molecules can be readilyattached to their surface for targeting to tissues. Finally,cost-effective manufacture of liposome-based pharmaceuticals, either ina liquid suspension or lyophilized product, has demonstrated theviability of this technology as an acceptable drug delivery system.

In some aspects, formulations associated with the invention might beselected for a class of naturally occurring or chemically synthesized ormodified saturated and unsaturated fatty acid residues. Fatty acidsmight exist in a form of triglycerides, diglycerides or individual fattyacids. In another embodiment, the use of well-validated mixtures offatty acids and/or fat emulsions currently used in pharmacology forparenteral nutrition may be utilized.

Liposome based formulations are widely used for oligonucleotidedelivery. However, most of commercially available lipid or liposomeformulations contain at least one positively charged lipid (cationiclipids). The presence of this positively charged lipid is believed to beessential for obtaining a high degree of oligonucleotide loading and forenhancing liposome fusogenic properties. Several methods have beenperformed and published to identify optimal positively charged lipidchemistries. However, the commercially available liposome formulationscontaining cationic lipids are characterized by a high level oftoxicity. In vivo limited therapeutic indexes have revealed thatliposome formulations containing positive charged lipids are associatedwith toxicity (i.e. elevation in liver enzymes) at concentrations onlyslightly higher than concentration required to achieve RNA silencing.

Nucleic acids associated with the invention can be hydrophobicallymodified and can be encompassed within neutral nanotransporters. Furtherdescription of neutral nanotransporters is incorporated by referencefrom PCT Application PCT/US2009/005251, filed on Sep. 22, 2009, andentitled “Neutral Nanotransporters.” Such particles enable quantitativeoligonucleotide incorporation into non-charged lipid mixtures. The lackof toxic levels of cationic lipids in such neutral nanotransportercompositions is an important feature.

As demonstrated in PCT/US2009/005251, oligonucleotides can effectivelybe incorporated into a lipid mixture that is free of cationic lipids andsuch a composition can effectively deliver a therapeutic oligonucleotideto a cell in a manner that it is functional. For example, a high levelof activity was observed when the fatty mixture was composed of aphosphatidylcholine base fatty acid and a sterol such as a cholesterol.For instance, one preferred formulation of neutral fatty mixture iscomposed of at least 20% of DOPC or DSPC and at least 20% of sterol suchas cholesterol. Even as low as 1:5 lipid to oligonucleotide ratio wasshown to be sufficient to get complete encapsulation of theoligonucleotide in a non-charged formulation.

The neutral nanotransporters compositions enable efficient loading ofoligonucleotide into neutral fat formulation. The composition includesan oligonucleotide that is modified in a manner such that thehydrophobicity of the molecule is increased (for example a hydrophobicmolecule is attached (covalently or no-covalently) to a hydrophobicmolecule on the oligonucleotide terminus or a non-terminal nucleotide,base, sugar, or backbone), the modified oligonucleotide being mixed witha neutral fat formulation (for example containing at least 25% ofcholesterol and 25% of DOPC or analogs thereof). A cargo molecule, suchas another lipid can also be included in the composition. Thiscomposition, where part of the formulation is built into theoligonucleotide itself, enables efficient encapsulation ofoligonucleotide in neutral lipid particles.

In some aspects, stable particles ranging in size from 50 to 140 nm canbe formed upon complexing of hydrophobic oligonucleotides with preferredformulations. It is interesting to mention that the formulation byitself typically does not form small particles, but rather, formsagglomerates, which are transformed into stable 50-120 nm particles uponaddition of the hydrophobic modified oligonucleotide.

The neutral nanotransporter compositions of the invention include ahydrophobic modified polynucleotide, a neutral fatty mixture, andoptionally a cargo molecule. A “hydrophobic modified polynucleotide” asused herein is a polynucleotide of the invention (i.e. sd-rxRNA) thathas at least one modification that renders the polynucleotide morehydrophobic than the polynucleotide was prior to modification. Themodification may be achieved by attaching (covalently or non-covalently)a hydrophobic molecule to the polynucleotide. In some instances thehydrophobic molecule is or includes a lipophilic group.

The term “lipophilic group” means a group that has a higher affinity forlipids than its affinity for water. Examples of lipophilic groupsinclude, but are not limited to, cholesterol, a cholesteryl or modifiedcholesteryl residue, adamantine, dihydrotesterone, long chain alkyl,long chain alkenyl, long chain alkynyl, olely-lithocholic, cholenic,oleoyl-cholenic, palmityl, heptadecyl, myrisityl, bile acids, cholicacid or taurocholic acid, deoxycholate, oleyl litocholic acid, oleoylcholenic acid, glycolipids, phospholipids, sphingolipids, isoprenoids,such as steroids, vitamins, such as vitamin E, fatty acids eithersaturated or unsaturated, fatty acid esters, such as triglycerides,pyrenes, porphyrines, Texaphyrine, adamantane, acridines, biotin,coumarin, fluorescein, rhodamine, Texas-Red, digoxygenin,dimethoxytrityl, t-butyldimethylsilyl, t-butyldiphenylsilyl, cyaninedyes (e.g. Cy3 or Cy5), Hoechst 33258 dye, psoralen, or ibuprofen. Thecholesterol moiety may be reduced (e.g. as in cholestan) or may besubstituted (e.g. by halogen). A combination of different lipophilicgroups in one molecule is also possible.

The hydrophobic molecule may be attached at various positions of thepolynucleotide. As described above, the hydrophobic molecule may belinked to the terminal residue of the polynucleotide such as the 3′ of5′-end of the polynucleotide. Alternatively, it may be linked to aninternal nucleotide or a nucleotide on a branch of the polynucleotide.The hydrophobic molecule may be attached, for instance to a 2′-positionof the nucleotide. The hydrophobic molecule may also be linked to theheterocyclic base, the sugar or the backbone of a nucleotide of thepolynucleotide.

The hydrophobic molecule may be connected to the polynucleotide by alinker moiety. Optionally the linker moiety is a non-nucleotidic linkermoiety. Non-nucleotidic linkers are e.g. abasic residues (dSpacer),oligoethyleneglycol, such as triethyleneglycol (spacer 9) orhexaethylenegylcol (spacer 18), or alkane-diol, such as butanediol. Thespacer units are preferably linked by phosphodiester or phosphorothioatebonds. The linker units may appear just once in the molecule or may beincorporated several times, e.g. via phosphodiester, phosphorothioate,methylphosphonate, or amide linkages.

Typical conjugation protocols involve the synthesis of polynucleotidesbearing an aminolinker at one or more positions of the sequence,however, a linker is not required. The amino group is then reacted withthe molecule being conjugated using appropriate coupling or activatingreagents. The conjugation reaction may be performed either with thepolynucleotide still bound to a solid support or following cleavage ofthe polynucleotide in solution phase. Purification of the modifiedpolynucleotide by HPLC typically results in a pure material.

In some embodiments the hydrophobic molecule is a sterol type conjugate,a PhytoSterol conjugate, cholesterol conjugate, sterol type conjugatewith altered side chain length, fatty acid conjugate, any otherhydrophobic group conjugate, and/or hydrophobic modifications of theinternal nucleoside, which provide sufficient hydrophobicity to beincorporated into micelles.

For purposes of the present invention, the term “sterols”, refers orsteroid alcohols are a subgroup of steroids with a hydroxyl group at the3-position of the A-ring. They are amphipathic lipids synthesized fromacetyl-coenzyme A via the HMG-CoA reductase pathway. The overallmolecule is quite flat. The hydroxyl group on the A ring is polar. Therest of the aliphatic chain is non-polar. Usually sterols are consideredto have an 8 carbon chain at position 17.

For purposes of the present invention, the term “sterol type molecules”,refers to steroid alcohols, which are similar in structure to sterols.The main difference is the structure of the ring and number of carbonsin a position 21 attached side chain.

For purposes of the present invention, the term “PhytoSterols” (alsocalled plant sterols) are a group of steroid alcohols, phytochemicalsnaturally occurring in plants. There are more than 200 different knownPhytoSterols

For purposes of the present invention, the term “Sterol side chain”refers to a chemical composition of a side chain attached at theposition 17 of sterol-type molecule. In a standard definition sterolsare limited to a 4 ring structure carrying a 8 carbon chain at position17. In this invention, the sterol type molecules with side chain longerand shorter than conventional are described. The side chain may branchedor contain double back bones.

Thus, sterols useful in the invention, for example, includecholesterols, as well as unique sterols in which position 17 hasattached side chain of 2-7 or longer than 9 carbons. In a particularembodiment, the length of the polycarbon tail is varied between 5 and 9carbons. Such conjugates may have significantly better in vive efficacy,in particular delivery to liver. These types of molecules are expectedto work at concentrations 5 to 9 fold lower then oligonucleotidesconjugated to conventional cholesterols.

Alternatively the polynucleotide may be bound to a protein, peptide orpositively charged chemical that functions as the hydrophobic molecule.The proteins may be selected from the group consisting of protamine,dsRNA binding domain, and arginine rich peptides. Exemplary positivelycharged chemicals include spermine, spermidine, cadaverine, andputrescine.

In another embodiment hydrophobic molecule conjugates may demonstrateeven higher efficacy when it is combined with optimal chemicalmodification patterns of the polynucleotide (as described herein indetail), containing but not limited to hydrophobic modifications,phosphorothioate modifications, and 2′ ribo modifications.

In another embodiment the sterol type molecule may be a naturallyoccurring PhytoSterols. The polycarbon chain may be longer than 9 andmay be linear, branched and/or contain double bonds. Some PhytoSterolcontaining polynucleotide conjugates may be significantly more potentand active in delivery of polynucleotides to various tissues. SomePhytoSterols may demonstrate tissue preference and thus be used as a wayto delivery RNAi specifically to particular tissues.

The hydrophobic modified polynucleotide is mixed with a neutral fattymixture to form a micelle. The neutral fatty acid mixture is a mixtureof fats that has a net neutral or slightly net negative charge at oraround physiological pH that can form a micelle with the hydrophobicmodified polynucleotide. For purposes of the present invention, the term“micelle” refers to a small nanoparticle formed by a mixture ofnon-charged fatty acids and phospholipids. The neutral fatty mixture mayinclude cationic lipids as long as they are present in an amount thatdoes not cause toxicity. In preferred embodiments the neutral fattymixture is free of cationic lipids. A mixture that is free of cationiclipids is one that has less than 1% and preferably 0% of the total lipidbeing cationic lipid. The term “cationic lipid” includes lipids andsynthetic lipids having a net positive charge at or around physiologicalpH. The term “anionic lipid” includes lipids and synthetic lipids havinga net negative charge at or around physiological pH.

The neutral fats bind to the oligonucleotides of the invention by astrong but non-covalent attraction (e.g., an electrostatic, van derWaals, pi-stacking, etc. interaction).

The neutral fat mixture may include formulations selected from a classof naturally occurring or chemically synthesized or modified saturatedand unsaturated fatty acid residues. Fatty acids might exist in a formof triglycerides, diglycerides or individual fatty acids. In anotherembodiment the use of well-validated mixtures of fatty acids and/or fatemulsions currently used in pharmacology for parenteral nutrition may beutilized.

The neutral fatty mixture is preferably a mixture of a choline basedfatty acid and a sterol. Choline based fatty acids include for instance,synthetic phosphocholine derivatives such as DDPC, DLPC, DMPC, DPPC,DSPC, DOPC, POPC, and DEPC. DOPC (chemical registry number 4235-95-4) isdioleoylphosphatidylcholine (also known asdielaidoylphosphatidylcholine, dioleoyl-PC, dioleoylphosphocholine,dioleoyl-sn-glycero-3-phosphocholine, dioleylphosphatidylcholine). DSPC(chemical registry number 816-94-4) is distearoylphosphatidylcholine(also known as 1,2-Distearoyl-sn-Glycero-3-phosphocholine).

The sterol in the neutral fatty mixture may be for instance cholesterol.The neutral fatty mixture may be made up completely of a choline basedfatty acid and a sterol or it may optionally include a cargo molecule.For instance, the neutral fatty mixture may have at least 20% or 25%fatty acid and 20% or 25% sterol.

For purposes of the present invention, the term “Fatty acids” relates toconventional description of fatty acid. They may exist as individualentities or in a form of two- and triglycerides. For purposes of thepresent invention, the term “fat emulsions” refers to safe fatformulations given intravenously to subjects who are unable to getenough fat in their diet. It is an emulsion of soy bean oil (or othernaturally occurring oils) and egg phospholipids. Fat emulsions are beingused for formulation of some insoluble anesthetics. In this disclosure,fat emulsions might be part of commercially available preparations likeIntralipid, Liposyn, Nutrilipid, modified commercial preparations, wherethey are enriched with particular fatty acids or fully denovo-formulated combinations of fatty acids and phospholipids.

In one embodiment, the cells to be contacted with an oligonucleotidecomposition of the invention are contacted with a mixture comprising theoligonucleotide and a mixture comprising a lipid, e.g., one of thelipids or lipid compositions described supra for between about 12 hoursto about 24 hours. In another embodiment, the cells to be contacted withan oligonucleotide composition are contacted with a mixture comprisingthe oligonucleotide and a mixture comprising a lipid, e.g., one of thelipids or lipid compositions described supra for between about 1 andabout five days. In one embodiment, the cells are contacted with amixture comprising a lipid and the oligonucleotide for between aboutthree days to as long as about 30 days. In another embodiment, a mixturecomprising a lipid is left in contact with the cells for at least aboutfive to about 20 days. In another embodiment, a mixture comprising alipid is left in contact with the cells for at least about seven toabout 15 days. 50%-60%/o of the formulation can optionally be any otherlipid or molecule. Such a lipid or molecule is referred to herein as acargo lipid or cargo molecule. Cargo molecules include but are notlimited to intralipid, small molecules, fusogenic peptides or lipids orother small molecules might be added to alter cellular uptake, endosomalrelease or tissue distribution properties. The ability to tolerate cargomolecules is important for modulation of properties of these particles,if such properties are desirable. For instance the presence of sometissue specific metabolites might drastically alter tissue distributionprofiles. For example use of Intralipid type formulation enriched inshorter or longer fatty chains with various degrees of saturationaffects tissue distribution profiles of these type of formulations (andtheir loads).

An example of a cargo lipid useful according to the invention is afusogenic lipid. For instance, the zwiterionic lipid DOPE (chemicalregistry number 4004-5-1, 1,2-Dioleoyl-sn-Glycero-3-phosphoethanolamine)is a preferred cargo lipid.

Intralipid may be comprised of the following composition: 1000 mLcontain: purified soybean oil 90 g, purified egg phospholipids 12 g,glycerol anhydrous 22 g, water for injection q.s. ad 1000 mL. pH isadjusted with sodium hydroxide to pH approximately 8. Energy content/L:4.6 MJ (190 kcal). Osmolality (approx.): 300 mOsm/kg water. In anotherembodiment fat emulsion is Liposyn that contains 5% safflower oil, 5%soybean oil, up to 1.2% egg phosphatides added as an emulsifier and 2.5%glycerin in water for injection. It may also contain sodium hydroxidefor pH adjustment. pH 8.0 (6.0-9.0). Liposyn has an osmolarity of 276 mOsmol/liter (actual).

Variation in the identity, amounts and ratios of cargo lipids affectsthe cellular uptake and tissue distribution characteristics of thesecompounds. For example, the length of lipid tails and level ofsaturability will affect differential uptake to liver, lung, fat andcardiomyocytes. Addition of special hydrophobic molecules like vitaminsor different forms of sterols can favor distribution to special tissueswhich are involved in the metabolism of particular compounds. In someembodiments, vitamin A or E is used. Complexes are formed at differentoligonucleotide concentrations, with higher concentrations favoring moreefficient complex formation.

In another embodiment, the fat emulsion is based on a mixture of lipids.Such lipids may include natural compounds, chemically synthesizedcompounds, purified fatty acids or any other lipids. In yet anotherembodiment the composition of fat emulsion is entirely artificial. In aparticular embodiment, the fat emulsion is more than 70% linoleic acid.In yet another particular embodiment the fat emulsion is at least 1% ofcardiolipin. Linoleic acid (LA) is an unsaturated omega-6 fatty acid. Itis a colorless liquid made of a carboxylic acid with an 18-carbon chainand two cis double bonds.

In yet another embodiment of the present invention, the alteration ofthe composition of the fat emulsion is used as a way to alter tissuedistribution of hydrophobicly modified polynucleotides. This methodologyprovides for the specific delivery of the polynucleotides to particulartissues.

In another embodiment the fat emulsions of the cargo molecule containmore than 70% of Linoleic acid (C18H32O2) and/or cardiolipin.

Fat emulsions, like intralipid have been used before as a deliveryformulation for some non-water soluble drugs (such as Propofol,re-formulated as Diprivan). Unique features of the present inventioninclude (a) the concept of combining modified polynucleotides with thehydrophobic compound(s), so it can be incorporated in the fat micellesand (b) mixing it with the fat emulsions to provide a reversiblecarrier. After injection into a blood stream, micelles usually bind toserum proteins, including albumin, HDL, LDL and other. This binding isreversible and eventually the fat is absorbed by cells. Thepolynucleotide, incorporated as a part of the micelle will then bedelivered closely to the surface of the cells. After that cellularuptake might be happening though variable mechanisms, including but notlimited to sterol type delivery.

Complexing Agents

Complexing agents bind to the oligonucleotides of the invention by astrong but non-covalent attraction (e.g., an electrostatic, van derWaals, pi-stacking, etc. interaction). In one embodiment,oligonucleotides of the invention can be complexed with a complexingagent to increase cellular uptake of oligonucleotides. An example of acomplexing agent includes cationic lipids. Cationic lipids can be usedto deliver oligonucleotides to cells. However, as discussed above,formulations free in cationic lipids are preferred in some embodiments.

The term “cationic lipid” includes lipids and synthetic lipids havingboth polar and non-polar domains and which are capable of beingpositively charged at or around physiological pH and which bind topolyanions, such as nucleic acids, and facilitate the delivery ofnucleic acids into cells. In general cationic lipids include saturatedand unsaturated alkyl and alicyclic ethers and esters of amines, amides,or derivatives thereof. Straight-chain and branched alkyl and alkenylgroups of cationic lipids can contain, e.g., from 1 to about 25 carbonatoms. Preferred straight chain or branched alkyl or alkene groups havesix or more carbon atoms. Alicyclic groups include cholesterol and othersteroid groups. Cationic lipids can be prepared with a variety ofcounterions (anions) including, e.g., Cl⁻, Br⁻, I⁻, F⁻, acetate,trifluoroacetate, sulfate, nitrite, and nitrate.

Examples of cationic lipids include polyethylenimine, polyamidoamine(PAMAM) starburst dendrimers, Lipofectin (a combination of DOTMA andDOPE), Lipofectase, LIPOFECTAMINE™ (e.g., LIPOFECTAMINE™ 2000), DOPE,Cytofectin (Gilead Sciences, Foster City, Calif.), and Eufectins (JBL,San Luis Obispo, Calif.). Exemplary cationic liposomes can be made fromN-[1l-(2,3-dioleoloxy)-propyl]-N,N,N-trimethylammonium chloride (DOTMA),N-[1-(2,3-dioleoloxy)-propyl]-N,N,N-trimethylammonium methylsulfate(DOTAP), 3β-[N—(N′,N′-dimethylaminoethane)carbamoyl]cholesterol(DC-Chol),2,3,-dioleyloxy-N-[2(sperminecarboxamido)ethyl]-N,N-dimethyl-1-propanaminiumtrifluoroacetate (DOSPA),1,2-dimyristyloxypropyl-3-dimethyl-hydroxyethyl ammonium bromide; anddimethyldioctadecylammonium bromide (DDAB). The cationic lipidN-(1-(2,3-dioleyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTMA),for example, was found to increase 1000-fold the antisense effect of aphosphorothioate oligonucleotide. (Vlassov et al., 1994, Biochimica etBiophysica Acta 1197:95-108). Oligonucleotides can also be complexedwith, e.g., poly (L-lysine) or avidin and lipids may, or may not, beincluded in this mixture, e.g., steryl-poly (L-lysine).

Cationic lipids have been used in the art to deliver oligonucleotides tocells (see, e.g., U.S. Pat. Nos. 5,855,910; 5,851,548; 5,830,430;5,780,053; 5,767,099; Lewis el al. 1996. Proc. Natl. Acad. Sci. USA93:3176; Hope et al. 1998. Molecular Membrane Biology 15:1). Other lipidcompositions which can be used to facilitate uptake of the instantoligonucleotides can be used in connection with the claimed methods. Inaddition to those listed supra, other lipid compositions are also knownin the art and include, e.g., those taught in U.S. Pat. No. 4,235,871;U.S. Pat. Nos. 4,501,728; 4,837,028; 4,737,323.

In one embodiment lipid compositions can further comprise agents, e.g.,viral proteins to enhance lipid-mediated transfections ofoligonucleotides (Kamata, et al., 1994. Nucl. Acids. Res. 22:536). Inanother embodiment, oligonucleotides are contacted with cells as part ofa composition comprising an oligonucleotide, a peptide, and a lipid astaught, e.g., in U.S. Pat. No. 5,736,392. Improved lipids have also beendescribed which are serum resistant (Lewis, et al., 1996. Proc. Natl.Acad. Sci. 93:3176). Cationic lipids and other complexing agents act toincrease the number of oligonucleotides carried into the cell throughendocytosis.

In another embodiment N-substituted glycine oligonucleotides (peptoids)can be used to optimize uptake of oligonucleotides. Peptoids have beenused to create cationic lipid-like compounds for transfection (Murphy,et al., 1998. Proc. Natl. Acad. Sci. 95:1517). Peptoids can besynthesized using standard methods (e.g., Zuckermann, R. N., et al.1992. J. Am. Chem. Soc. 114:10646; Zuckermann, R. N., et al. 1992. Int.J. Peptide Protein Res. 40:497). Combinations of cationic lipids andpeptoids, liptoids, can also be used to optimize uptake of the subjectoligonucleotides (Hunag, et al., 1998. Chemistry and Biology. 5:345).Liptoids can be synthesized by elaborating peptoid oligonucleotides andcoupling the amino terminal submonomer to a lipid via its amino group(Hunag, et al., 1998. Chemistry and Biology. 5:345).

It is known in the art that positively charged amino acids can be usedfor creating highly active cationic lipids (Lewis et al. 1996. Proc.Natl. Acad. Sci. U.S.A. 93:3176). In one embodiment, a composition fordelivering oligonucleotides of the invention comprises a number ofarginine, lysine, histidine or ornithine residues linked to a lipophilicmoiety (see e.g., U.S. Pat. No. 5,777,153).

In another embodiment, a composition for delivering oligonucleotides ofthe invention comprises a peptide having from between about one to aboutfour basic residues. These basic residues can be located, e.g., on theamino terminal, C-terminal, or internal region of the peptide. Familiesof amino acid residues having similar side chains have been defined inthe art. These families include amino acids with basic side chains(e.g., lysine, arginine, histidine), acidic side chains (e.g., asparticacid, glutamic acid), uncharged polar side chains (e.g., glycine (canalso be considered non-polar), asparagine, glutamine, serine, threonine,tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine,leucine, isoleucine, proline, phenylalanine, methionine, tryptophan),beta-branched side chains (e.g., threonine, valine, isoleucine) andaromatic side chains (e.g., tyrosine, phenylalanine, tryptophan,histidine). Apart from the basic amino acids, a majority or all of theother residues of the peptide can be selected from the non-basic aminoacids, e.g., amino acids other than lysine, arginine, or histidine.Preferably a preponderance of neutral amino acids with long neutral sidechains are used.

In one embodiment, a composition for delivering oligonucleotides of theinvention comprises a natural or synthetic polypeptide having one ormore gamma carboxyglutamic acid residues, or γ-Gla residues. These gammacarboxyglutamic acid residues may enable the polypeptide to bind to eachother and to membrane surfaces. In other words, a polypeptide having aseries of γ-Gla may be used as a general delivery modality that helps anRNAi construct to stick to whatever membrane to which it comes incontact. This may at least slow RNAi constructs from being cleared fromthe blood stream and enhance their chance of homing to the target.

The gamma carboxyglutamic acid residues may exist in natural proteins(for example, prothrombin has 10γ-Gla residues). Alternatively, they canbe introduced into the purified, recombinantly produced, or chemicallysynthesized polypeptides by carboxylation using, for example, a vitaminK-dependent carboxylase. The gamma carboxyglutamic acid residues may beconsecutive or non-consecutive, and the total number and location ofsuch gamma carboxyglutamic acid residues in the polypeptide can beregulated/fine-tuned to achieve different levels of “stickiness” of thepolypeptide.

In one embodiment, the cells to be contacted with an oligonucleotidecomposition of the invention are contacted with a mixture comprising theoligonucleotide and a mixture comprising a lipid, e.g., one of thelipids or lipid compositions described supra for between about 12 hoursto about 24 hours. In another embodiment, the cells to be contacted withan oligonucleotide composition are contacted with a mixture comprisingthe oligonucleotide and a mixture comprising a lipid, e.g., one of thelipids or lipid compositions described supra for between about 1 andabout five days. In one embodiment, the cells are contacted with amixture comprising a lipid and the oligonucleotide for between aboutthree days to as long as about 30 days. In another embodiment, a mixturecomprising a lipid is left in contact with the cells for at least aboutfive to about 20 days. In another embodiment, a mixture comprising alipid is left in contact with the cells for at least about seven toabout 15 days.

For example, in one embodiment, an oligonucleotide composition can becontacted with cells in the presence of a lipid such as cytofectin CS orGSV (available from Glen Research: Sterling, Va.), GS3815, GS2888 forprolonged incubation periods as described herein.

In one embodiment, the incubation of the cells with the mixturecomprising a lipid and an oligonucleotide composition does not reducethe viability of the cells. Preferably, after the transfection periodthe cells are substantially viable. In one embodiment, aftertransfection, the cells are between at least about 70% and at leastabout 100% viable. In another embodiment, the cells are between at leastabout 80% and at least about 95% viable. In yet another embodiment, thecells are between at least about 85% and at least about 90% viable.

In one embodiment, oligonucleotides are modified by attaching a peptidesequence that transports the oligonucleotide into a cell, referred toherein as a “transporting peptide.” In one embodiment, the compositionincludes an oligonucleotide which is complementary to a target nucleicacid molecule encoding the protein, and a covalently attachedtransporting peptide.

The language “transporting peptide” includes an amino acid sequence thatfacilitates the transport of an oligonucleotide into a cell. Exemplarypeptides which facilitate the transport of the moieties to which theyare linked into cells are known in the art, and include, e.g., HIV TATtranscription factor, lactoferrin, Herpes VP22 protein, and fibroblastgrowth factor 2 (Pooga et al. 1998. Nature Biotechnology. 16:857; andDerossi et al. 1998. Trends in Cell Biology. 8:84; Elliott and O'Hare.1997. Cell 88:223).

Oligonucleotides can be attached to the transporting peptide using knowntechniques, e.g., (Prochiantz, A. 1996. Curr. Opin. Neurobiol. 6:629;Derossi et al. 1998. Trends Cell Biol. 8:84; Troy et al. 1996. J.Neurosci. 16:253), Vives et al. 1997. J. Biol. Chem. 272:16010). Forexample, in one embodiment, oligonucleotides bearing an activated thiolgroup are linked via that thiol group to a cysteine present in atransport peptide (e.g., to the cysteine present in the 3 turn betweenthe second and the third helix of the antennapedia homeodomain astaught, e.g., in Derossi et al. 1998. Trends Cell Biol. 8:84;Prochiantz. 1996. Current Opinion in Neurobiol. 6:629; Allinquant et al.1995. J Cell Biol. 128:919). In another embodiment, a Boc-Cys-(Npys)OHgroup can be coupled to the transport peptide as the last (N-terminal)amino acid and an oligonucleotide bearing an SH group can be coupled tothe peptide (Troy et al. 1996. J. Neurosci. 16:253).

In one embodiment, a linking group can be attached to a nucleomonomerand the transporting peptide can be covalently attached to the linker.In one embodiment, a linker can function as both an attachment site fora transporting peptide and can provide stability against nucleases.Examples of suitable linkers include substituted or unsubstituted C₁-C₂₀alkyl chains, C₂-C₂₀ alkenyl chains, C₂-C₂₀ alkynyl chains, peptides,and heteroatoms (e.g., S, O, NH, etc.). Other exemplary linkers includebifinctional crosslinking agents such assulfosuccinimidyl-4-(maleimidophenyl)-butyrate (SMPB) (see, e.g., Smithet al. Biochem J 1991.276: 417-2).

In one embodiment, oligonucleotides of the invention are synthesized asmolecular conjugates which utilize receptor-mediated endocytoticmechanisms for delivering genes into cells (see, e.g., Bunnell et al.1992. Somatic Cell and Molecular Genetics. 18:559, and the referencescited therein).

Targeting Agents

The delivery of oligonucleotides can also be improved by targeting theoligonucleotides to a cellular receptor. The targeting moieties can beconjugated to the oligonucleotides or attached to a carrier group (i.e.,poly(L-lysine) or liposomes) linked to the oligonucleotides. This methodis well suited to cells that display specific receptor-mediatedendocytosis.

For instance, oligonucleotide conjugates to 6-phosphomannosylatedproteins are internalized 20-fold more efficiently by cells expressingmannose 6-phosphate specific receptors than free oligonucleotides. Theoligonucleotides may also be coupled to a ligand for a cellular receptorusing a biodegradable linker. In another example, the delivery constructis mannosylated streptavidin which forms a tight complex withbiotinylated oligonucleotides. Mannosylated streptavidin was found toincrease 20-fold the internalization of biotinylated oligonucleotides.(Vlassov et al. 1994. Biochimica et Biophysica Acta 1197:95-108).

In addition specific ligands can be conjugated to the polylysinecomponent of polylysine-based delivery systems. For example,transferrin-polylysine, adenovirus-polylysine, and influenza virushemagglutinin HA-2 N-terminal fusogenic peptides-polylysine conjugatesgreatly enhance receptor-mediated DNA delivery in eucaryotic cells.

Mannosylated glycoprotein conjugated to poly(L-lysine) in aveolarmacrophages has been employed to enhance the cellular uptake ofoligonucleotides. Liang et al. 1999. Pharmazie 54:559-566.

Because malignant cells have an increased need for essential nutrientssuch as folic acid and transferrin, these nutrients can be used totarget oligonucleotides to cancerous cells. For example, when folic acidis linked to poly(L-lysine) enhanced oligonucleotide uptake is seen inpromyelocytic leukaemia (HL-60) cells and human melanoma (M-14) cells.Ginobbi et al. 1997. Anticancer Res. 17:29. In another example,liposomes coated with maleylated bovine serum albumin, folic acid, orferric protoporphyrin IX, show enhanced cellular uptake ofoligonucleotides in murine macrophages, KB cells, and 2.2.15 humanhepatoma cells. Liang et al. 1999. Pharmazie 54:559-566.

Liposomes naturally accumulate in the liver, spleen, andreticuloendothelial system (so-called, passive targeting). By couplingliposomes to various ligands such as antibodies are protein A, they canbe actively targeted to specific cell populations. For example, proteinA-bearing liposomes may be pretreated with H-2K specific antibodieswhich are targeted to the mouse major histocompatibility complex-encodedH-2K protein expressed on L cells. (Vlassov el al. 1994. Biochimica etBiophysica Acta 1197:95-108).

Other in vitro and/or in vivo delivery of RNAi reagents are known in theart, and can be used to deliver the subject RNAi constructs. See, forexample, U.S. patent application publications 20080152661, 20080112916,20080107694, 20080038296, 20070231392, 20060240093, 20060178327,20060008910, 20050265957, 20050064595, 20050042227, 20050037496,20050026286, 20040162235, 20040072785, 20040063654, 20030157030, WO2008/036825, WO04/065601, and AU2004206255B2, just to name a few (allincorporated by reference).

Administration

The optimal course of administration or delivery of the oligonucleotidesmay vary depending upon the desired result and/or on the subject to betreated. As used herein “administration” refers to contacting cells witholigonucleotides and can be performed in vitro or in vivo. The dosage ofoligonucleotides may be adjusted to optimally reduce expression of aprotein translated from a target nucleic acid molecule, e.g., asmeasured by a readout of RNA stability or by a therapeutic response,without undue experimentation.

For example, expression of the protein encoded by the nucleic acidtarget can be measured to determine whether or not the dosage regimenneeds to be adjusted accordingly. In addition, an increase or decreasein RNA or protein levels in a cell or produced by a cell can be measuredusing any art recognized technique. By determining whether transcriptionhas been decreased, the effectiveness of the oligonucleotide in inducingthe cleavage of a target RNA can be determined.

Any of the above-described oligonucleotide compositions can be usedalone or in conjunction with a pharmaceutically acceptable carrier. Asused herein, “pharmaceutically acceptable carrier” includes appropriatesolvents, dispersion media, coatings, antibacterial and antifungalagents, isotonic and absorption delaying agents, and the like. The useof such media and agents for pharmaceutical active substances is wellknown in the art. Except insofar as any conventional media or agent isincompatible with the active ingredient, it can be used in thetherapeutic compositions. Supplementary active ingredients can also beincorporated into the compositions.

In some embodiments, the disclosure relates to a composition (e.g.,pharmaceutical composition) comprising an oligonucleotide (e.g., anisolated double stranded nucleic acid molecule). In some embodiments,the composition comprises an additional therapeutic agent. Non-limitingexamples of additional therapeutic agents include but are not limited tonucleic acids (e.g., sd-rxRNA, etc.), small molecules (e.g., smallmolecules useful for treating cancer, neurodegenerative diseases,infectious diseases, autoimmune diseases, etc.), peptides (e.g.,peptides useful for treating cancer, neurodegenerative diseases,infectious diseases, autoimmune diseases, etc.), and polypeptides (e.g.,antibodies useful for treating cancer, neurodegenerative diseases,infectious diseases, autoimmune diseases, etc.). Compositions of thedisclosure can have, in some embodiments, 2, 3, 4, 5, 6, 7, 8, 9, 10, ormore additional therapeutic agents. In some embodiments, a compositioncomprises more than 10 additional therapeutic agents.

Oligonucleotides may be incorporated into liposomes or liposomesmodified with polyethylene glycol or admixed with cationic lipids forparenteral administration. Incorporation of additional substances intothe liposome, for example, antibodies reactive against membrane proteinsfound on specific target cells, can help target the oligonucleotides tospecific cell types.

With respect to in vivo applications, the formulations of the presentinvention can be administered to a patient in a variety of forms adaptedto the chosen route of administration, e.g., parenterally, orally, orintraperitoneally, infusion, intrathecal delivery, parenchymal delivery,intravenous delivery or direct injection into the brain or spinal cord.

Aspects of the invention relate to delivery of nucleic acid molecules,such as sd-rxRNA or sd-rxRNA variants to the nervous system. Forexample, an sd-rxRNA or sd-rxRNA variant can be delivered to the brainor to the spinal cord. Any appropriate delivery mechanism for deliveringan sd-rxRNA variant to the brain or spinal cord can be applied. In someembodiments, delivery to the brain or spinal cord occurs by infusion,intrathecal delivery, parenchymal delivery, intravenous delivery ordirect injection into the brain or spinal cord. In some embodiments, ansd-rxRNA or an sd-rxRNA variant is delivered to a specific region of thebrain. An sd-rxRNA or an sd-rxRNA variant can be modified or formulatedappropriately to pass the blood-brain barrier. In other embodiments, ansd-rxRNA or an sd-rxRNA variant is administered in such a way that itdoes not need to cross the blood-brain barrier. In some embodiments, thesd-rxRNA or an sd-rxRNA variant is delivered by a pump or cathetersystem into the brain or spinal cord. Examples of such delivery areincorporated by reference from U.S. Pat. No. 6,093,180 (Elsberry).Techniques for infusing drugs into the brain are also incorporated byreference from U.S. Pat. No. 5,814,014 (Elsberry et al.).

In some embodiments, nucleic acids are administered by parenteraladministration, which includes administration by the following routes:intravenous; intramuscular; interstitially; intraarterially;subcutaneous; intra ocular; intrasynovial; trans epithelial, includingtransdermal; pulmonary via inhalation; ophthalmic; sublingual andbuccal; topically, including ophthalmic; dermal; ocular; rectal: andnasal inhalation via insufflation. In some embodiments, the sd-rxRNAmolecules are administered by intradermal injection or subcutaneously.

Pharmaceutical preparations for administration can include aqueoussolutions of the active compounds in water-soluble or water-dispersibleform. In addition, suspensions of the active compounds as appropriateoily injection suspensions may be administered. Suitable lipophilicsolvents or vehicles include fatty oils, for example, sesame oil, orsynthetic fatty acid esters, for example, ethyl oleate or triglycerides.Aqueous injection suspensions may contain substances which increase theviscosity of the suspension include, for example, sodium carboxymethylcellulose, sorbitol, or dextran, optionally, the suspension may alsocontain stabilizers. The oligonucleotides of the invention can beformulated in liquid solutions, preferably in physiologically compatiblebuffers such as Hank's solution or Ringer's solution. In addition, theoligonucleotides may be formulated in solid form and redissolved orsuspended immediately prior to use. Lyophilized forms are also includedin the invention.

Pharmaceutical preparations for administration can also includetransdermal patches, ointments, lotions, creams, gels, drops, sprays,suppositories, liquids and powders. In addition, conventionalpharmaceutical carriers, aqueous, powder or oily bases, or thickenersmay be used in pharmaceutical preparations for topical administration.

Pharmaceutical preparations for oral administration can also includepowders or granules, suspensions or solutions in water or non-aqueousmedia, capsules, sachets or tablets. In addition, thickeners, flavoringagents, diluents, emulsifiers, dispersing aids, or binders may be usedin pharmaceutical preparations for oral administration.

For transmucosal or transdermal administration, penetrants appropriateto the barrier to be permeated are used in the formulation. Suchpenetrants are known in the art, and include, for example, fortransmucosal administration bile salts and fusidic acid derivatives, anddetergents. Transmucosal administration may be through nasal sprays orusing suppositories. For oral administration, the oligonucleotides areformulated into conventional oral administration forms such as capsules,tablets, and tonics. For topical administration, the oligonucleotides ofthe invention are formulated into ointments, salves, gels, or creams asknown in the art.

Drug delivery vehicles can be chosen e.g., for in vitro, for systemic,or for topical administration. These vehicles can be designed to serveas a slow release reservoir or to deliver their contents directly to thetarget cell. An advantage of using some direct delivery drug vehicles isthat multiple molecules are delivered per uptake. Such vehicles havebeen shown to increase the circulation half-life of drugs that wouldotherwise be rapidly cleared from the blood stream. Some examples ofsuch specialized drug delivery vehicles which fall into this categoryare liposomes, hydrogels, cyclodextrins, biodegradable nanocapsules, andbioadhesive microspheres.

The described oligonucleotides may be administered systemically to asubject. Systemic absorption refers to the entry of drugs into the bloodstream followed by distribution throughout the entire body.Administration routes which lead to systemic absorption include:intravenous, subcutaneous, intraperitoneal, and intranasal. Each ofthese administration routes delivers the oligonucleotide to accessiblediseased cells. Following subcutaneous administration, the therapeuticagent drains into local lymph nodes and proceeds through the lymphaticnetwork into the circulation. The rate of entry into the circulation hasbeen shown to be a function of molecular weight or size. The use of aliposome or other drug carrier localizes the oligonucleotide at thelymph node. The oligonucleotide can be modified to diffuse into thecell, or the liposome can directly participate in the delivery of eitherthe unmodified or modified oligonucleotide into the cell.

The chosen method of delivery will result in entry into cells. In someembodiments, preferred delivery methods include liposomes (10-400 nm),hydrogels, controlled-release polymers, and other pharmaceuticallyapplicable vehicles, and microinjection or electroporation (for ex vivotreatments).

The pharmaceutical preparations of the present invention may be preparedand formulated as emulsions. Emulsions are usually heterogeneous systemsof one liquid dispersed in another in the form of droplets usuallyexceeding 0.1 μm in diameter. The emulsions of the present invention maycontain excipients such as emulsifiers, stabilizers, dyes, fats, oils,waxes, fatty acids, fatty alcohols, fatty esters, humectants,hydrophilic colloids, preservatives, and anti-oxidants may also bepresent in emulsions as needed. These excipients may be present as asolution in either the aqueous phase, oily phase or itself as a separatephase.

Examples of naturally occurring emulsifiers that may be used in emulsionformulations of the present invention include lanolin, beeswax,phosphatides, lecithin and acacia. Finely divided solids have also beenused as good emulsifiers especially in combination with surfactants andin viscous preparations. Examples of finely divided solids that may beused as emulsifiers include polar inorganic solids, such as heavy metalhydroxides, nonswelling clays such as bentonite, attapulgite, hectorite,kaolin, montrnorillonite, colloidal aluminum silicate and colloidalmagnesium aluminum silicate, pigments and nonpolar solids such as carbonor glyceryl tristearate.

Examples of preservatives that may be included in the emulsionformulations include methyl paraben, propyl paraben, quaternary ammoniumsalts, benzalkonium chloride, esters of p-hydroxybenzoic acid, and boricacid. Examples of antioxidants that may be included in the emulsionformulations include free radical scavengers such as tocopherols, alkylgallates, butylated hydroxyanisole, butylated hydroxytoluene, orreducing agents such as ascorbic acid and sodium metabisulfite, andantioxidant synergists such as citric acid, tartaric acid, and lecithin.

In one embodiment, the compositions of oligonucleotides are formulatedas microemulsions. A microemulsion is a system of water, oil andamphiphile which is a single optically isotropic and thermodynamicallystable liquid solution. Typically microemulsions are prepared by firstdispersing an oil in an aqueous surfactant solution and then adding asufficient amount of a 4th component, generally an intermediatechain-length alcohol to form a transparent system.

Surfactants that may be used in the preparation of microemulsionsinclude, but are not limited to, ionic surfactants, non-ionicsurfactants, Brij 96, polyoxyethylene oleyl ethers, polyglycerol fattyacid esters, tetraglycerol monolaurate (ML310), tetraglycerol monooleate(M0310), hexaglycerol monooleate (P0310), hexaglycerol pentaoleate(P0500), decaglycerol monocaprate (MCA750), decaglycerol monooleate(M0750), decaglycerol sequioleate (S0750), decaglycerol decaoleate(DA0750), alone or in combination with cosurfactants. The cosurfactant,usually a short-chain alcohol such as ethanol, 1-propanol, and1-butanol, serves to increase the interfacial fluidity by penetratinginto the surfactant film and consequently creating a disordered filmbecause of the void space generated among surfactant molecules.

Microemulsions may, however, be prepared without the use ofcosurfactants and alcohol-free self-emulsifying microemulsion systemsare known in the art. The aqueous phase may typically be, but is notlimited to, water, an aqueous solution of the drug, glycerol, PEG300,PEG400, polyglycerols, propylene glycols, and derivatives of ethyleneglycol. The oil phase may include, but is not limited to, materials suchas Captex 300, Captex 355, Capmul MCM, fatty acid esters, medium chain(C₈-C₁₂) mono, di, and tri-glycerides, polyoxyethylated glyceryl fattyacid esters, fatty alcohols, polyglycolized glycerides, saturatedpolyglycolized C₈-C₁₀ glycerides, vegetable oils and silicone oil.

Microemulsions are particularly of interest from the standpoint of drugsolubilization and the enhanced absorption of drugs. Lipid basedmicroemulsions (both oil/water and water/oil) have been proposed toenhance the oral bioavailability of drugs.

Microemulsions offer improved drug solubilization, protection of drugfrom enzymatic hydrolysis, possible enhancement of drug absorption dueto surfactant-induced alterations in membrane fluidity and permeability,ease of preparation, ease of oral administration over solid dosageforms, improved clinical potency, and decreased toxicity (Constantinideset al., Pharmaceutical Research, 1994, 11:1385; Ho et al., J. Pharm.Sci., 1996, 85:138-143). Microemulsions have also been effective in thetransdermal delivery of active components in both cosmetic andpharmaceutical applications. It is expected that the microemulsioncompositions and formulations of the present invention will facilitatethe increased systemic absorption of oligonucleotides from thegastrointestinal tract, as well as improve the local cellular uptake ofoligonucleotides within the gastrointestinal tract, vagina, buccalcavity and other areas of administration.

In an embodiment, the present invention employs various penetrationenhancers to affect the efficient delivery of nucleic acids,particularly oligonucleotides, to the skin of animals. Evennon-lipophilic drugs may cross cell membranes if the membrane to becrossed is treated with a penetration enhancer. In addition toincreasing the diffusion of non-lipophilic drugs across cell membranes,penetration enhancers also act to enhance the permeability of lipophilicdrugs.

Five categories of penetration enhancers that may be used in the presentinvention include: surfactants, fatty acids, bile salts, chelatingagents, and non-chelating non-surfactants. Other agents may be utilizedto enhance the penetration of the administered oligonucleotides include:glycols such as ethylene glycol and propylene glycol, pyrrols such as2-15 pyrrol, azones, and terpenes such as limonene, and menthone.

The oligonucleotides, especially in lipid formulations, can also beadministered by coating a medical device, for example, a catheter, suchas an angioplasty balloon catheter, with a cationic lipid formulation.Coating may be achieved, for example, by dipping the medical device intoa lipid formulation or a mixture of a lipid formulation and a suitablesolvent, for example, an aqueous-based buffer, an aqueous solvent,ethanol, methylene chloride, chloroform and the like. An amount of theformulation will naturally adhere to the surface of the device which issubsequently administered to a patient, as appropriate. Alternatively, alyophilized mixture of a lipid formulation may be specifically bound tothe surface of the device. Such binding techniques are described, forexample, in K. Ishihara et al., Journal of Biomedical MaterialsResearch, Vol. 27, pp. 1309-1314 (1993), the disclosures of which areincorporated herein by reference in their entirety.

The useful dosage to be administered and the particular mode ofadministration will vary depending upon such factors as the cell type,or for in vivo use, the age, weight and the particular animal and regionthereof to be treated, the particular oligonucleotide and deliverymethod used, the therapeutic or diagnostic use contemplated, and theform of the formulation, for example, suspension, emulsion, micelle orliposome, as will be readily apparent to those skilled in the art.Typically, dosage is administered at lower levels and increased untilthe desired effect is achieved. When lipids are used to deliver theoligonucleotides, the amount of lipid compound that is administered canvary and generally depends upon the amount of oligonucleotide agentbeing administered. For example, the weight ratio of lipid compound tooligonucleotide agent is preferably from about 1:1 to about 15:1, with aweight ratio of about 5:1 to about 10:1 being more preferred. Generally,the amount of cationic lipid compound which is administered will varyfrom between about 0.1 milligram (mg) to about 1 gram (g). By way ofgeneral guidance, typically between about 0.1 mg and about 10 mg of theparticular oligonucleotide agent, and about 1 mg to about 100 mg of thelipid compositions, each per kilogram of patient body weight, isadministered, although higher and lower amounts can be used.

The agents of the invention are administered to subjects or contactedwith cells in a biologically compatible form suitable for pharmaceuticaladministration. By “biologically compatible form suitable foradministration” is meant that the oligonucleotide is administered in aform in which any toxic effects are outweighed by the therapeuticeffects of the oligonucleotide. In one embodiment, oligonucleotides canbe administered to subjects. Examples of subjects include mammals, e.g.,humans and other primates; cows, pigs, horses, and farming(agricultural) animals; dogs, cats, and other domesticated pets; mice,rats, and transgenic non-human animals.

Administration of an active amount of an oligonucleotide of the presentinvention is defined as an amount effective, at dosages and for periodsof time necessary to achieve the desired result. For example, an activeamount of an oligonucleotide may vary according to factors such as thetype of cell, the oligonucleotide used, and for in vivo uses the diseasestate, age, sex, and weight of the individual, and the ability of theoligonucleotide to elicit a desired response in the individual.Establishment of therapeutic levels of oligonucleotides within the cellis dependent upon the rates of uptake and efflux or degradation.Decreasing the degree of degradation prolongs the intracellularhalf-life of the oligonucleotide. Thus, chemically-modifiedoligonucleotides, e.g., with modification of the phosphate backbone, mayrequire different dosing.

The exact dosage of an oligonucleotide and number of doses administeredwill depend upon the data generated experimentally and in clinicaltrials. Several factors such as the desired effect, the deliveryvehicle, disease indication, and the route of administration, willaffect the dosage. Dosages can be readily determined by one of ordinaryskill in the art and formulated into the subject pharmaceuticalcompositions. Preferably, the duration of treatment will extend at leastthrough the course of the disease symptoms.

Dosage regimens may be adjusted to provide the optimum therapeuticresponse. For example, the oligonucleotide may be repeatedlyadministered, e.g., several doses may be administered daily or the dosemay be proportionally reduced as indicated by the exigencies of thetherapeutic situation. One of ordinary skill in the art will readily beable to determine appropriate doses and schedules of administration ofthe subject oligonucleotides, whether the oligonucleotides are to beadministered to cells or to subjects.

Administration of sd-rxRNAs can be optimized through testing of dosingregimens.

In some embodiments, a single administration is sufficient. To furtherprolong the effect of the administered sd-rxRNA, the sd-rxRNA can beadministered in a slow-release formulation or device, as would befamiliar to one of ordinary skill in the art. The hydrophobic nature ofsd-rxRNA compounds can enable use of a wide variety of polymers, some ofwhich are not compatible with conventional oligonucleotide delivery.

In other embodiments, the sd-rxRNA is administered multiple times. Insome instances it is administered daily, bi-weekly, weekly, every twoweeks, every three weeks, monthly, every two months, every three months,every four months, every five months, every six months or lessfrequently than every six months. In some instances, it is administeredmultiple times per day, week, month and/or year. For example, it can beadministered approximately every hour, 2 hours, 3 hours, 4 hours, 5hours, 6 hours, 7 hours, 8 hours, 9 hours 10 hours, 12 hours or morethan twelve hours. It can be administered 1, 2, 3, 4, 5, 6, 7, 8, 9, 10or more than 10 times per day.

Aspects of the invention relate to administering sd-rxRNA molecules to asubject. In some instances the subject is a patient and administeringthe sd-rxRNA molecule involves administering the sd-rxRNA molecule in adoctor's office.

In some embodiments, more than one sd-rxRNA molecule is administeredsimultaneously. For example a composition may be administered thatcontains 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more than 10 differentsd-rxRNA molecules. In certain embodiments, a composition comprises 2 or3 different sd-rxRNA molecules. When a composition comprises more thanone sd-rxRNA, the sd-rxRNA molecules within the composition can bedirected to the same gene or to different genes.

In some instances, the effective amount of sd-rxRNA that is delivered isat least approximately 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, 20, 21,22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32,33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50,51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61,62, 63, 64, 65, 66, 67, 68,69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86,87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100 or more than 100mg/kg including any intermediate values.

In some instances, the effective amount of sd-rxRNA that is delivered isat least approximately 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60,65, 70, 75, 80, 85, 90, 95, 100, 125, 150, 200, 250, 300, 350, 400, 450,500, 550, 600, 650, 700, 750, 800, 850, 900, 950 or more than 950 μgincluding any intermediate values.

sd-rxRNA molecules administered through methods described herein can beeffectively targeted to all the cell types in the nervous system.

Various modalities of introducing nucleic acids into a subject (e.g., acell of a subject) are contemplated by the disclosure. For example,nucleic acids (e.g., a solution containing the nucleic acids) can beinjected into a subject (e.g., injected into a cell) or a subject (e.g.,a cell) can be bombarded by particles covered by the nucleic acids. Insome embodiments, the cell or organism is soaked in a solution of thenucleic acid. In some embodiments, a nucleic acid is introduced into anorganism or cell by electroporation of cell membranes in the presence ofthe nucleic acid. In some embodiments, a viral construct comprising thenucleic acid is packaged into a viral particle and accomplishesintroduction of the nucleic acid into the cell and transcription ofnucleic acid. Further examples of modalities for introducing nucleicacids into a subject (e.g., a cell of a subject) include but are notlimited to lipid-mediated carrier transport, chemical-mediated transport(e.g., calcium phosphate), etc.

Nucleic acids can be introduced with additional components. For example,in some embodiments, the nucleic acid is introduced with a componentthat enhances nucleic acid uptake by the cell. In some embodiments, thenucleic acid is introduced with a component that inhibits annealing ofsingle strands. In some embodiments, the nucleic acid is introduced witha component that stabilizes the nucleic acid molecule, or other-wiseincreases inhibition of the target gene.

Nucleic acid may be directly introduced into the cell (i.e.,intracellularly); or introduced extracellularly into a cavity,interstitial space, into the circulation of an organism, introducedorally, or may be introduced by bathing a cell or organism in a solutioncontaining the nucleic acid. Vascular or extravascular circulation, theblood or lymph system, and the cerebrospinal fluid are sites where thenucleic acid may be introduced.

In some embodiments, the cell with the target gene may be derived fromany organism. In some embodiments, the cell with the target gene may becontained in (e.g., housed by, or present within) any organism. Forexample, the organism may a plant, animal, protozoan, bacterium, virus,or fungus. The plant may be a monocot, dicot or gymnosperm; the animalmay be a vertebrate or invertebrate. Preferred microbes are those usedin agriculture or by industry, and those that are pathogenic for plantsor animals.

Alternatively, vectors, e.g., transgenes encoding a siRNA of theinvention can be engineered into a host cell or transgenic animal usingart recognized techniques.

A further preferred use for the agents of the present invention (orvectors or transgenes encoding same) is a functional analysis to becarried out in eukaryotic cells, or eukaryotic non-human organisms,preferably mammalian cells or organisms and most preferably human cells,e.g. cell lines such as HeLa or 293 or rodents, e.g. rats and mice. Byadministering a suitable priming agent/RNAi agent which is sufficientlycomplementary to a target mRNA sequence to direct target-specific RNAinterference, a specific knockout or knockdown phenotype can be obtainedin a target cell, e.g. in cell culture or in a target organism.

Thus, a further subject matter of the invention is a eukaryotic cell ora eukaryotic non-human organism exhibiting a target gene-specificknockout or knockdown phenotype comprising a fully or at least partiallydeficient expression of at least one endogenous target gene wherein saidcell or organism is transfected with at least one vector comprising DNAencoding an RNAi agent capable of inhibiting the expression of thetarget gene. It should be noted that the present invention allows atarget-specific knockout or knockdown of several different endogenousgenes due to the specificity of the RNAi agent.

Gene-specific knockout or knockdown phenotypes of cells or non-humanorganisms, particularly of human cells or non-human mammals may be usedin analytic to procedures, e.g. in the functional and/or phenotypicalanalysis of complex physiological processes such as analysis of geneexpression profiles and/or proteomes. Preferably the analysis is carriedout by high throughput methods using oligonucleotide based chips.

Therapeutic Use

By inhibiting the expression of a gene, the oligonucleotide compositionsof the present invention can be used to treat any disease involving theexpression of a protein, such as neurodegenerative disease.

Aspects of the invention relate to the use of nucleic acid molecules,such as sd-rxRNA or sd-rxRNA variants sd-rxRNA in treatment of disordersaffecting the nervous system. In some embodiments, the sd-rxRNA is usedto treat a neurodegenerative disorder. As used herein, the term“neurodegenerative disorder” refers to disorders, diseases or conditionsthat are caused by the deterioration of cell and tissue components ofthe nervous system. Some non-limiting examples of neurodegenerativedisorders include stroke, Alzheimer's disease, Parkinson's disease,Huntington's disease, Peri ventricular leukomalacia (PVL), amyotrophiclateral sclerosis (ALS, “Lou Gehrig's disease”),ALS-Parkinson's-Dementia complex of Guam, Friedrich's Ataxia, Wilson'sdisease, multiple sclerosis, cerebral palsy, progressive supranuclearpalsy (Steel-Richardson syndrome), bulbar and pseudobulbar palsy,diabetic retinopathy, multi-infarct dementia, macular degeneration,Pick's disease, diffuse Lewy body disease, prion diseases such asCreutzfeldt-Jakob, Gerstmann-Straussler-Scheinker disease, Kuru andfatal familial insomnia, primary lateral sclerosis, degenerativeataxias, Machado-Joseph disease/spinocerebellar ataxia type 3 andolivopontocerebellar degenerations, spinal and spinobulbar muscularatrophy (Kennedy's disease), familial spastic paraplegia,Wohlfart-Kugelberg-Welander disease, Tay-Sach's disease, multisystemdegeneration (Shy-Drager syndrome), Gilles De La Tourette's disease,familial dysautonomia (Riley-Day syndrome), Kugelberg-Welander disease,subacute sclerosing panencephalitis, Werdnig-Hoffinann disease,synucleinopathies (including multiple system atrophy), Sandhoff disease,cortical basal degeneration, spastic paraparesis, primary progressiveaphasia, progressive multifocal leukoencephalopathy, striatonigraldegeneration, familial spastic disease, chronic epileptic conditionsassociated with neurodegeneration, Binswanger's disease, and dementia(including all underlying etiologies of dementia).

In some embodiments, the disorder is Parkinson's disease Huntington's orALS. In certain embodiments, the disorder is ALS and the sd-rxRNA orsd-rxRNA variant targets SOD1, a superoxide dismutase.

Neurodegenerative disorders may also be the result of a brain injury ortrauma including that which is caused by a stroke, an injury to the heador spinal cord, or acute ischemic injury. Ischemic injuries refer toconditions that arise when the brain receives insufficient blood flow.In some embodiments, injury to the brain or nervous system can resultfrom a traumatic injury, or could be the result of infection, radiation,chemical or toxic damage. Injury within the brain and nervous system,which may be diffuse or localized, includes an intracranial or intravertebral lesion or hemorrhage, cerebral ischemia or infarctionincluding embolic occlusion and thrombotic occlusion, perinatalhypoxic-ischemic injury, whiplash, shaken infant syndrome, reperfusionfollowing acute ischemia, or cardiac arrest. In one embodiment, in vitrotreatment of cells with oligonucleotides can be used for ex vivo therapyof cells removed from a subject (e.g., for treatment of leukemia orviral infection) or for treatment of cells which did not originate inthe subject, but are to be administered to the subject (e.g., toeliminate transplantation antigen expression on cells to be transplantedinto a subject). In addition, in vitro treatment of cells can be used innontherapeutic settings, e.g., to evaluate gene function, to study generegulation and protein synthesis or to evaluate improvements made tooligonucleotides designed to modulate gene expression or proteinsynthesis. In vivo treatment of cells can be useful in certain clinicalsettings where it is desirable to inhibit the expression of a protein.There are numerous medical conditions for which antisense therapy isreported to be suitable (see, e.g., U.S. Pat. No. 5,830,653) as well asrespiratory syncytial virus infection (WO 95/22,553) influenza virus (WO94/23,028), and malignancies (WO 94/08,003). Other examples of clinicaluses of antisense sequences are reviewed, e.g., in Glaser. 1996. GeneticEngineering News 16:1. Exemplary targets for cleavage byoligonucleotides include, e.g., protein kinase Ca, ICAM-1, c-raf kinase,p53, c-myb, and the bcr/abl fusion gene found in chronic myelogenousleukemia.

The subject nucleic acids can be used in RNAi-based therapy in anyanimal having RNAi pathway, such as human, non-human primate, non-humanmammal, non-human vertebrates, rodents (mice, rats, hamsters, rabbits,etc.), domestic livestock animals, pets (cats, dogs, etc.), Xenopus,fish, insects (Drosophila, etc.), and worms (C. elegans), etc.

The invention provides methods for preventing in a subject, a disease orcondition associated with an aberrant or unwanted target gene expressionor activity, by administering to the subject a therapeutic agent (e.g.,a RNAi agent or vector or transgene encoding same). If appropriate,subjects are first treated with a priming agent so as to be moreresponsive to the subsequent RNAi therapy. Subjects at risk for adisease which is caused or contributed to by aberrant or unwanted targetgene expression or activity can be identified by, for example, any or acombination of diagnostic or prognostic assays as described herein.Administration of a prophylactic agent can occur prior to themanifestation of symptoms characteristic of the target gene aberrancy,such that a disease or disorder is prevented or, alternatively, delayedin its progression. Depending on the type of target gene aberrancy, forexample, a target gene, target gene agonist or target gene antagonistagent can be used for treating the subject.

In another aspect, the invention pertains to methods of modulatingtarget gene expression, protein expression or activity for therapeuticpurposes. Accordingly, in an exemplary embodiment, the modulatory methodof the invention involves contacting a cell capable of expressing targetgene with a therapeutic agent of the invention that is specific for thetarget gene or protein (e.g., is specific for the mRNA encoded by saidgene or specifying the amino acid sequence of said protein) such thatexpression or one or more of the activities of target protein ismodulated. These modulatory methods can be performed in vitro (e.g., byculturing the cell with the agent), in vivo (e.g., by administering theagent to a subject), or ex vivo. Typically, subjects are first treatedwith a priming agent so as to be more responsive to the subsequent RNAitherapy. As such, the present invention provides methods of treating anindividual afflicted with a disease or disorder characterized byaberrant or unwanted expression or activity of a target gene polypeptideor nucleic acid molecule. Inhibition of target gene activity isdesirable in situations in which target gene is abnormally unregulatedand/or in which decreased target gene activity is likely to have abeneficial effect.

The therapeutic agents of the invention can be administered toindividuals to treat (prophylactically or therapeutically) disordersassociated with aberrant or unwanted target gene activity. Inconjunction with such treatment, pharmacogenomics (i.e., the study ofthe relationship between an individual's genotype and that individual'sresponse to a foreign compound or drug) may be considered. Differencesin metabolism of therapeutics can lead to severe toxicity or therapeuticfailure by altering the relation between dose and blood concentration ofthe pharmacologically active drug. Thus, a physician or clinician mayconsider applying knowledge obtained in relevant pharmacogenomicsstudies in determining whether to administer a therapeutic agent as wellas tailoring the dosage and/or therapeutic regimen of treatment with atherapeutic agent. Pharmacogenomics deals with clinically significanthereditary variations in the response to drugs due to altered drugdisposition and abnormal action in affected persons. See, for example,Eichelbaum, M. et al. (1996) Clin. Exp. Pharmacol. Physiol. 23(10-11):983-985 and Linder, M. W. et al. (1997) Clin. Chem. 43(2):254-266

The present invention is further illustrated by the following Examples,which in no way should be construed as further limiting. The entirecontents of all of the references (including literature references,issued patents, published patent applications, and co pending patentapplications) cited throughout this application are hereby expresslyincorporated by reference.

EXAMPLES Example 1: Identification of SOD1-Targeting sd-rxRNA Variants

sd-rxRNA variants targeting SOD1 were designed, synthesized and screenedin vitro to determine the ability of the sd-rxRNA variant to reducetarget gene mRNA levels. The sd-rxRNA variants were tested for activityin HeLa cells (human cervical carcinoma cell line, 10,000 cells/well, 96well plate). HeLa cells were treated with varying concentrations of apanel of SOD1-targeting sd-rxRNAs variants or non-targeting control inserum containing media. Concentrations tested were 5, 1 and 0.1 μM. Thenon-targeting control sd-rxRNA is of similar structure to theSOD1-targeting sd-rxRNA variants and contains similar stabilizingmodifications throughout both strands. Forty eight hours postadministration, cells were lysed and mRNA levels determined by theQuantigene branched DNA assay according to the manufacture's protocolusing gene-specific probes (Affymetrix, Santa Clara, Calif.). Exemplarysense and antisense sequences are presented in Tables 1 and 2 below.FIGS. 1 and 2 demonstrate that the SOD1-targeting sd-rxRNA variantssignificantly reduce target gene mRNA levels in vitro in HeLa cells.Data were normalized to a house keeping gene (PPIB) and graphed withrespect to the non-targeting control. Error bars represent the standarddeviation from the mean of biological duplicates. Sequencescorresponding to FIGS. 1 and 2 can be found in Tables 3 and 4,respectively.

TABLE 1 Exemplary sense oligonucleotides SEQ Oligo Gene ID Sense Numbersymbol Sense sequence NO: Sense Chemistry Backbone Notes 25634 SOD1GAGAGGCAUGUU  1 DY547mm0m00m0 oooooooooo A m0mmm sso 25635 SOD1GAGAGGCAUGUU  2 DY547mm0m00m0 oooooooooo A m0mmm sso 25636 SOD1GAGAGGCAUGUU  3 DY547mm0m00m0 oooooooooo A m0mmm sso 25637 SOD1GAGAGGCAUGUU  4 DY547mm0m00m0 sssssssssssso A m0mmm 25600 SOD1GAGAGGCAUGUU  5 mm0000m0m0mm sssssssssssso A m 25638 SOD1 GAGAGGCAUGUU 6 DY547mm0m00m0 sssssssssssso A m0mmm 25643 SOD1 GAGAGGCAUGUU  7DY547mm0m00m0 oooooooooo A m0mmm sso 25644 SOD1 GAGAGGCAUGUU  8DY547mm0m00m0 sssssssssssso A m0mmm 25645 SOD1 GAGAGGCAUGUU  9DY547mm0m00m0 oooooooooo A m0mmm sso 25652 SOD1 GAGAGGCAUGUU 10DY547mm0000m0m sssssssssssso A 0mmm 25568 SOD1 AGGZGGAAAZGAA 11DY547mm0m00000 sssssssssssso Z = octyl, x = 5 m0mm methyl C, Y = 5methyl U 25569 SOD1 AGGYGGAAAZGAA 12 DY547mm0m00000 sssssssssssso Z =octyl, x = 5 m0mm methyl C, Y = 5 methyl U 25570 SOD1 AGGZGGAAAYGAA 13DY547mm0m00000 sssssssssssso Z = octyl, x = 5 m0mm methyl C, Y = 5methyl U 25571 SOD1 AGGYGGAAAYGAA 14 DY547mm0m00000 sssssssssssso Z =octyl, x = 5 m0mm methyl C, Y = 5 methyl U 25572 SOD1 AGGUGGAAAUGA 15DY547mm0m00000 sssssssssssso Z = octyl, x = 5 A m0mm methyl C, Y = 5methyl U 25573 SOD1 AGGUGGAAAUGA 16 DY547mm0m00000 sssssssssssso Z =octyl, x = 5 A m0mm methyl C, Y = 5 methyl U 25574 SOD1 AGGUGGAAAUGA 17DY547mm0m00000 sssssssssssso Z = octyl, x = 5 A m0mm methyl C, Y = 5methyl U 25575 SOD1 AGGUGGAAAUGA 18 DY547mm0m00000 sssssssssssso Z =octyl, x = 5 A m0mm methyl C, Y = 5 methyl U 25576 SOD1 AGGUGGAAAUGA 19DY547mm0m00000 sssssssssssso x = 5 methyl A m0mm C, Y = 5 methyl U 25578SOD1 AGGYGGAAAZGAA 20 DY547mm0m00000 sssssssssssso Z = thiophene, m0mmx = 5 methyl C, Y = 5 methyl U 25579 SOD1 AGGZGGAAAYGAA 21DY547mm0m00000 sssssssssssso Z = thiophene, m0mm x = 5 methyl C, Y  =5 methyl U 25580 SOD1 AGGYGGAAAYGAA 22 DY547mm0m00000 sssssssssssso Z =thiophene, m0mm x = 5 methyl C, Y = 5 methyl U 25584 SOD1 AGGUGGAAAUGA23 DY547mm0m00000 sssssssssssso Z = thiophene, A m0mm x = 5 methylC, Y = 5 methyl U 25585 SOD1 AGGUGGAAAUGA 24 DY547mm0m00000sssssssssssso Z = thiophene, A m0mm x = 5 methyl C, Y = 5 methyl U 25586SOD1 AGGZGGAAAZGAA 25 DY547mmOd00000d sssssssssssso Z = isobutyl, 0mmx = 5 methyl C, Y = 5 methyl U 25587 SOD1 AGGYGGAAAZGAA 26DY547mm0m00000 sssssssssssso Z = isobutyl, d0mm x = 5 methyl C, Y =5 methyl U 25588 SOD1 AGGZGGAAAYGAA 27 DY547mmOd00000 sssssssssssso Z =isobutyl, m0mm x = 5 methyl C, Y = 5 methyl U 25589 SOD1 AGGUGGAAAUGA 28DY547mm0m00000 sssssssssssso Z = isobutyl, A m0mm x = 5 methyl C, Y =5 methyl U 25590 SOD1 AGGUGGAAAUGA 29 DY547mm0m00000 sssssssssssso Z =isobutyl, A m0mm x = 5 methyl C, Y = 5 methyl U 24560 SOD1 AGGUGGAAAUGA30 DY547mm0m00000 sssssssssssso A m0mm 25634 no F1 SOD1 GAGAGGCAUGUU 31mm0m00m0m0mm oooooooooo label A m sso 25635 no F1 SOD1 GAGAGGCAUGUU 32mm0m00m0m0mm oooooooooo label A m sso 25636 no F1 SOD1 GAGAGGCAUGUU 33mm0m00m0m0mm oooooooooo label A m sso 25637 no F1 SOD1 GAGAGGCAUGUU 34mm0m00m0m0mm sssssssssssso label A m 25638 no F1 SOD1 GAGAGGCAUGUU 35mm0m00m0m0mm sssssssssssso label A m 25643 no F1 SOD1 GAGAGGCAUGUU 36mm0m00m0m0mm oooooooooo label A m sso 25644 no F1 SOD1 GAGAGGCAUGUU 37mm0m00m0m0mm sssssssssssso label A m 25645 no F1 SOD1 GAGAGGCAUGUU 38mm0m00m0m0mm oooooooooo label A m sso 25652 no F1 SOD1 GAGAGGCAUGUU 39mm0000m0m0mm sssssssssssso label A m 25568 no F1 SOD1 AGGZGGAAAZGAA 40mm0m00000m0mm sssssssssssso Z = octyl, x = 5 label methyl C, Y = 5methyl U 25569 no F1 SOD1 AGGYGGAAAZGAA 41 mm0m00000m0mm ssssssssssssoZ = octyl, x = 5 label methyl C, Y = 5 methyl U 25570 no F1 SOD1AGGZGGAAAYGAA 42 mm0m00000m0mm sssssssssssso Z = octyl, x = 5 labelmethyl C, Y = 5 methyl U 25571 no F1 SOD1 AGGYGGAAAYGAA 43 mm0m00000m0mmsssssssssssso Z = octyl, x = 5 label methyl C, Y = 5 methyl U25572 no F1 SOD1 AGGUGGAAAUGA 44 mm0m00000m0mm sssssssssssso Z =octyl, x = 5 label A methyl C, Y = 5 methyl U 25573 no F1 SOD1AGGUGGAAAUGA 45 mm0m00000m0mm sssssssssssso Z = octyl, x = 5 label Amethyl C, Y = 5 methyl U 25574 no F1 SOD1 AGGUGGAAAUGA 46 mm0m00000m0mmsssssssssssso Z = octyl, x = 5 label A methyl C, Y = 5 methyl U25575 no F1 SOD1 AGGUGGAAAUGA 47 mm0m00000m0mm sssssssssssso Z =octyl, x = 5 label A methyl C, Y = 5 methyl U 25576 no F1 SOD1AGGUGGAAAUGA 48 mm0m00000m0mm sssssssssssso x = 5 methyl label A C, Y =5 methyl U 25578 no F1 SOD1 AGGYGGAAAZGAA 49 mm0m00000m0mm ssssssssssssoZ = thiophene, label x = 5 methyl C, Y = 5 methyl U 25579 no F1 SOD1AGGZGGAAAYGAA 50 mm0m00000m0mm sssssssssssso Z = thiophene, label x =5 methyl C, Y = 5 methyl U 25580 no F1 SOD1 AGGYGGAAAYGAA 51mm0m00000m0mm sssssssssssso Z = thiophene, label x = 5 methyl C, Y =5 methyl U 25584 no F1 SOD1 AGGUGGAAAUGA 52 mm0m00000m0mm ssssssssssssoZ = thiophene, label A x = 5 methyl C, Y = 5 methyl U 25585 no F1 SOD1AGGUGGAAAUGA 53 mm0m00000m0mm sssssssssssso Z = thiophene, label A x =5 methyl C, Y = 5 methyl U 25586 no F1 SOD1 AGGZGGAAAZGAA 54mm0d00000d0mm sssssssssssso Z = isobutyl, label x = 5 methyl C, Y =5 methyl U 25587 no F1 SOD1 AGGYGGAAAZGAA 55 mm0m00000d0mm ssssssssssssoZ = isobutyl, label x = 5 methyl C, Y = 5 methyl U 25588 no F1 SOD1AGGZGGAAAYGAA 56 mm0d00000m0mm sssssssssssso Z = isobutyl, label x =5 methyl C, Y = 5 methyl U 25589 no F1 SOD1 AGGUGGAAAUGA 57mm0m00000m0mm sssssssssssso Z = isobutyl, label A x = 5 methyl C, Y =5 methyl U 25590 no F1 SOD1 AGGUGGAAAUGA 58 mm0m00000m0mm ssssssssssssoZ = isobutyl, label A x = 5 methyl C, Y = 5 methyl U 24560 no F1 SOD1AGGUGGAAAUGA 59 mm0m00000m0mm sssssssssssso label A

TABLE 2 Exemplary antisense oligonucleotides Oligo Antisense SEQ IDAntiSense Number sequence NO: AntiSense Chemistry Backbone Notes 25634UAACAUGCCUCUC  60 Pm00f0f0fffff0fff0fff0 ssssssssssssssssssss UUCAUCCU o25635 UAACAUGCCUCUC  61 Pm00f0fOfffffffffOfff0 oooooooooooossss UUCAUCCUsssso 25636 UAACAUGCCUCUC  62 Pm00f0f0fffffffff0f0 oooooooooooossssUUCAUC sso 25637 UAACAUGCCUCUC  63 Pm00f0f0fffffffff0fff0oooooooooooossss UUCAUCCU sssso 25600  UAACAUGCCUCUC  64Pm00f0f0ffffffffff0fff0 ssssssssssssssssssss UUCAUCCU o 25638UAACAUGCCUCUC  65 Pm00f0f0fffffffff00 oooooooooooossss UUCAUCCU sssso25643 UAACAUGCCUCUC  66 Pm00f0fOfffffffff0f0 sssssssssssssssssso UUCAUC25644 UAACAUGCCUCUC  67 Pm00f0f0fffffffff0f0 sssssssssssssssssso UUCAUC25645 UAACAUGCCUCUC  68 Pm00f0f0fffffffff0fff0 ssssssssssssssssssssUUCAUCCU o 25652 UAACAUGCCUCUC  69 Pm00f0f0ffffffffff0fff0ssssssssssssssssssss UUCAUCCU o 25568 UUCAUUUCCACCU  70Pmff0fffff0fmmmm0m ssssssssssssssssssss Z = octyl, x = 5 UUGCCCAA mf00 omethyl C, Y = 5 methyl U 25569 UUCAUUUCCACCU  71 Pmff0fffff0fmmmmOmssssssssssssssssssss Z = octyl, x = 5 UUGCCCAA mf00 o methyl C, Y = 5methyl U 25570 UUCAUUUCCACCU  72 Pmff0fffff0fmmmm0m ssssssssssssssssssssZ = octyl, x = 5 UUGCCCAA mf00 o methyl C, Y = 5 methyl U 25571UUCAUUUCCACCU  73 Pmff0fffff0fmmmm0m ssssssssssssssssssss Z = octyl, x =5 UUGCCCAA mf00 o methyl C, Y = 5 methyl U 25572 YZXAYYZXXAXXZYY  74Pmff0fffff0fmmmm0m ssssssssssssssssssss Z = octyl, x = 5 GXXXAA mf00 omethyl C, Y = 5 methyl U 25573 YYXAYYZXXAXXZYY  75 Pmff0fffff0fmmmm0mssssssssssssssssssss Z = octyl, x = 5 GXXXAA mf00 o methyl C, Y = 5methyl U 25574 YZXAYYYXXAXXZYY  76 Pmff0fffff0fmmmm0mssssssssssssssssssss Z = octyl, x = 5 GXXXAA mf00 o methyl C, Y = 5methyl U 25575 YZXAYYZXXAXXYYY 77 Pmff0fffff0fmmmm0mssssssssssssssssssss Z = octyl, x = 5 GXXXAA mf00 o methyl C, Y = 5methyl U 25576 YYXAYYYXXAXXYYY  78 Pmff0fffff0fmmmm0mssssssssssssssssssss x = 5 methyl C, GXXXAA mf00 o Y = 5 methyl U 25578UUCAUUUCCACCU  79 Pmff0fffff0fmmmm0m ssssssssssssssssssss Z = thiophene,UUGCCCAA mf00 o x = 5 methyl C, Y = 5 methyl U 25579 UUCAUUUCCACCU  80Pmff0fffff0fmmmm0m ssssssssssssssssssss Z = thiophene, UUGCCCAA mf00 ox = 5 methyl C, Y = 5 methyl U 25580 UUCAUUUCCACCU  81Pmff0fffff0fmmmm0m ssssssssssssssssssss Z = thiophene, UUGCCCAA mf00 ox = 5 methyl C, Y = 5 methyl U 25584 YZXAYYZXXAXXYYY  82Pmff0fffff0fmmmm0m ssssssssssssssssssss Z = thiophene, GXXXAA mf00 o x =5 methyl C, Y = 5 methyl U 25585 YYXAYYYXXAXXYYY  83 Pmff0fffff0fmmmm0mssssssssssssssssssss Z = thiophene, GXXXAA mf00 o x = 5 methyl C, Y =5 methyl U 25586 UUCAUUUCCACCU  84 Pmff0fffff0fmmmm0mssssssssssssssssssss Z = isobutyl, x = UUGCCCAA mf00 o 5 methyl C, Y =5 methyl U 25587 UUCAUUUCCACCU  85 Pmff0fffff0fmmmm0mssssssssssssssssssss Z = isobutyl, x = UUGCCCAA mf00 o 5 methyl C, Y =5 methyl U 25588 UUCAUUUCCACCU  86 Pmff0fffff0fmmmm0mssssssssssssssssssss Z = isobutyl, x = UUGCCCAA mf00 o 5 methyl C, Y =5 methyl U 25589 YZXAYYZXXAXXZYY  87 Pmdf0ffdff0fmdmm0mssssssssssssssssssss Z = isobutyl, x = GXXXAA mf00 o 5 methyl C, Y =5 methyl U 25590 YYXAYYZXXAXXZYY  88 Pmff0ffdff0fmdmm0mssssssssssssssssssss Z = isobutyl, x = GXXXAA mf00 o 5 methyl C, Y =5 methyl U 24560 UUCAUUUCCACCU  89 Pmff0fffff0fmmmm0mssssssssssssssssssss UUGCCCAA mf00 o 25634 no UAACAUGCCUCUC  90Pm00f0f0fffff0fff0fff0 ssssssssssssssssssss F1 label UUCAUCCU o 25635 noUAACAUGCCUCUC  91 Pm00f0f0fffffffff0fff0 oooooooooooossss F1 labelUUCAUCCU sssso 25636 no UAACAUGCCUCUC  92 Pm00f0f0fffffffff0f0oooooooooooossss F1 label UUCAUC sso 25637 no UAACAUGCCUCUC  93Pm00f0f0fffffffff0fff0 oooooooooooossss F1 label UUCAUCCU sssso 25638 noUAACAUGCCUCUC  94 Pm00f0fOfffffffff00 oooooooooooossss F1 label UUCAUCCUsssso 25643 no UAACAUGCCUCUC  95 Pm00f0f0fffffffff0f0sssssssssssssssssso F1 label UUCAUC 25644 no UAACAUGCCUCUC  96Pm00f0f0fffffffff0f0 sssssssssssssssssso F1 label UUCAUC 25645 noUAACAUGCCUCUC  97 Pm00f0f0fffffffff0fff0 ssssssssssssssssssss F1 labelUUCAUCCU o 25652 no UAACAUGCCUCUC  98 Pm00f0f0fffff0fff0fff0ssssssssssssssssssss F1 label UUCAUCCU o 25568 no UUCAUUUCCACCU  99Pmff0fffff0fmmmm0m ssssssssssssssssssss Z = octyl, x = 5 F1 labelUUGCCCAA mf00 o methyl C, Y = 5 methyl U 25569 no UUCAUUUCCACCU 100Pmff0fffff0fmmmm0m ssssssssssssssssssss Z = octyl, x = 5 F1 labelUUGCCCAA mf00 o methyl C, Y = 5 methyl U 25570 no UUCAUUUCCACCU 101Pmff0fffff0fmmmm0m ssssssssssssssssssss Z = octyl, x = 5 F1 labelUUGCCCAA mf00 o methyl C, Y = 5 methyl U 25571 no UUCAUUUCCACCU 102Pmff0fffff0fmmmm0m ssssssssssssssssssss Z = octyl, x = 5 F1 labelUUGCCCAA mf00 o methyl C, Y = 5 methyl U 25572 no YZXAYYZXXAXXZYY 103Pmff0fffff0fmmmm0m ssssssssssssssssssss Z = octyl, x = 5 F1 label GXXXAAmf00 o methyl C, Y = 5 methyl U 25573 no YYXAYYZXXAXXZYY 104Pmff0fffff0fmmmm0m ssssssssssssssssssss Z = octyl, x = 5 F1 label GXXXAAmf00 o methyl C, Y = 5 methyl U 25574 no YZXAYYYXXAXXZYY 105Pmff0fffff0fmmmm0m ssssssssssssssssssss Z = octyl, x = 5 F1 label GXXXAAmf00 o methyl C, Y = 5 methyl U 25575 no YZXAYYZXXAXXYYY 106Pmff0fffff0fmmmm0m ssssssssssssssssssss Z = octyl, x = 5 F1 label GXXXAAmf00 o methyl C, Y = 5 methyl U 25576 no YYXAYYYXXAXXYYY 107Pmff0fffff0fmmmm0m ssssssssssssssssssss x = 5 methyl C, F1 label GXXXAAmf00 o Y = 5 methyl U 25578 no UUCAUUUCCACCU 108 Pmff0fffff0fmmmm0mssssssssssssssssssss Z = thiophene, F1 label UUGCCCAA mf00 o x =5 methyl C, Y = 5 methyl U 25579 no UUCAUUUCCACCU 109 Pmff0fffff0fmmmm0mssssssssssssssssssss Z = thiophene, F1 label UUGCCCAA mf00 o x =5 methyl C, Y = 5 methyl U 25580 no UUCAUUUCCACCU 110 Pmff0fffff0fmmmm0mssssssssssssssssssss Z = thiophene, F1 label UUGCCCAA mf00 o x =5 methyl C, Y = 5 methyl U 25584 no YZXAYYZXXAXXYYY 111Pmff0fffff0fmmmm0m ssssssssssssssssssss Z = thiophene, F1 label GXXXAAmf00 o x = 5 methyl C, Y = 5 methyl U 25585 no YYXAYYYXXAXXYYY 112Pmff0fffff0fmmmm0m ssssssssssssssssssss Z = thiophene, F1 label GXXXAAmf00 o x = 5 methyl C, Y = 5 methyl U 25586 no UUCAUUUCCACCU 113Pmff0fffff0fmmmm0m ssssssssssssssssssss Z = isobutyl, x = F1 labelUUGCCCAA mf00 o 5 methyl C, Y = 5 methyl U 25587 no UUCAUUUCCACCU 114Pmff0fffff0fmmmm0m ssssssssssssssssssss Z = isobutyl, x = F1 labelUUGCCCAA mf00 o 5 methyl C, Y = 5 methyl U 25588 no UUCAUUUCCACCU 115Pmff0fffff0fmmmm0m ssssssssssssssssssss Z = isobutyl, x = F1 labelUUGCCCAA mf00 o 5 methyl C, Y = 5 methyl U 25589 no YZXAYYZXXAXXZYY 116Pmdf0ffdff0fmdmm0m ssssssssssssssssssss Z = isobutyl, x = F1 labelGXXXAA mf00 o 5 methyl C, Y = 5 methyl U 25590 no YYXAYYZXXAXXZYY 117Pmff0ffdff0fmdmm0m ssssssssssssssssssss Z = isobutyl, x = F1 labelGXXXAA mf00 o 5 methyl C, Y = 5 methyl U 24560 no UUCAUUUCCACCU 118Pmff0fffff0fmmmm0m ssssssssssssssssssss F1 label UUGCCCAA mf00 o Key forTables 1 and 2: f = 2′fluoro M = 2′Ome P = 5′ phosphate s =phosphorothioate linkage o = phosphodiester linkage DY547 = DY547 dye d= deoxyribose

The human SOD1 sequence is represented by GenBank accession numberNM_000454.4

(SEQ ID NO: 119) listed below. Multiple mutations of the SOD1 sequencehave been identified (Rosen et al (1993) Nature; Deng et al (1993)Science, De Belleroche et al. (1995) J Med Genet.; Orrel et al (1997) JNeurol.; Cudkowicz (1997) Ann. Neurol.; and Anderson et al (1995) NatureGenet.) and can also be targeted utilizing the sequences outlined inthis application:

(SEQ ID NO: 119) GTTTGGGGCCAGAGTGGGCGAGGCGCGGAGGTCTGGCCTATAAAGTAGTCGCGGAGACGGGGTGCTGGTTTGCGTCGTAGTCTCCTGCAGCGTCTGGGGTTTCCGTTGCAGTCCTCGGAACCAGGACCTCGGCGTGGCCTAGCGAGTTATGGCGACGAAGGCCGTGTGCGTGCTGAAGGGCGACGGCCCAGTGCAGGGCATCATCAATTTCGAGCAGAAGGAAAGTAATGGACCAGTGAAGGTGTGGGGAAGCATTAAAGGACTGACTGAAGGCCTGCATGGATTCCATGTTCATGAGTTTGGAGATAATACAGCAGGCTGTACCAGTGCAGGTCCTCACTTTAATCCTCTATCCAGAAAACACGGTGGGCCAAAGGATGAAGAGAGGCATGTTGGAGACTTGGGCAATGTGACTGCTGACAAAGATGGTGTGGCCGATGTGTCTATTGAAGATTCTGTGATCTCACTCTCAGGAGACCATTGCATCATTGGCCGCACACTGGTGGTCCATGAAAAAGCAGATGACTTGGGCAAAGGTGGAAAGAAGAAAGTACAAAGACAGGAAACGCTGGAAGTCGTTTGGCTTGTGGTGTAATTGGGATCGCCCAATAAACATTCCCTTGGATGTAGTCTGAGGCCCCTTAACTCATCTGTTATCCTGCTAGCTGTAGAAATGTATCCTGATAAACATTAAACACTGTAATCTTAAAAGTGTAATTGTGTGACTTTTTCAGAGTTGCTTTAAAGTACCTGTAGTGAGAAACTGATTTATGATCACTTGGAAGATTTGTATAGTTTTATAAAACTCAGTTAAAATGTCTGTTTCAATGACCTGTATTTTGCCAGACTTAAATCACAGATGGGTATTAAACTTGTCAGAATTTCTTTGTCATTCAAGCCTGTGAATAAAAACCCTGTATGGCACTTATTATGAGGCTATTAAAAGAATCCAAATTCAAACTAAAAAAAAAAAAAAAAAA

TABLE 3Reducing Phosphorothioate Content Results in Active ps-rxRNA Variants withReduced Cellular Toxicity in vitro (sequences corresponding to FIG. 1)SEQ ID Total Oligo ID NO: # PS 25634 PSDY547.mG.mA. G.mA. G. G.mC. A.mU. G.mU*mU*mA 120 22 GSP.mU* A* A*fC* A*fU* G*fC*fC*fU*fC*fU* C*fU*fU*fC* A*fU*fC*fC* U 12125635 PS DY547.mG.mA. G.mA. G. G.mC. A.mU. G.mU*mU*mA 122 10 GSP.mU. A. A.fC. A.fU. GfC.fC.fU.fC.fU.fC*fU*fU.fC* A*fU*fC*fC* U 12325636 PS DY547.mG.mA. G.mA. G. G.mC. A.mU. G.mU*mU*mA 124  8 GSP.mU. A. A.fC. A.fU. G.fC.fC.fU.fC.fU. C*fU*fU*fC* A*fU* C 125 25637 PSDY547.mG*mA* G*mA* G* G*mC*A*mU* G*mU*mU*mA 126 20 GS 25600 PSmG*mA* G* A* G* G*mC* A*mU* G*mU*mU*mA 127 32 (Parent  GSP.mU* A* A*fC* A*fU* G*fC*fC*fU*fC*fU* C*fU*fU*fC* A*fU*fC*fC* U 128ps-rxRNA)

TABLE 4Reducing Phosphorothioate Content Results in Active ps-rxRNA Variants withReduced Cellular Toxicity in vitro (sequences corresponding to FIG. 2)SEQ ID Total  Oligo ID NO: # PS 25638 PsDY547.mG*mA* G*mA* G* G*mC* A*mU* G*mU*mU*mA 129 18 GSP.mU. A. A.fC. A.fU. G.fC.fC.fU.fC.fU.fC*fU*fU*fC* A*fU*fC*fC* U 13025643 PS DY547.mG.mA. G.mA. G. G.mC. A.mU. G.mU*mU*mA 131 20 GSP.mU* A* A*fC* A*fU* G*fC*fC*fU*fC*fU*fC*fU*fU*fC* A*fU* C 132 25644 PSDY547.mG*mA* G*mA* G* G*mC* A*mU* G*mU*mU*mA 133 30 GSP.mU* A* A*fC* A*fU* G*fC*fC*fU*fC*fU*fC*fU*fU*fC* A*fU* C 134 25645 PSDY547.mG.mA. G.mA. G. G.mC. A.mU. G.mU*mU*mA 135 22 GSP.mU* A* A*fC* A*fU* G*fC*fC*fU*fC*fU*fC*fU*fU*fC* A*fU*fC*fC* 136 U25600 PS mG*mA* G* A* G* G*mC* A*mU* G*mU*mU*mA 137 32 (Parent GSP.mU* A* A*fC* A*fU* G*fC*fC*fU*fC*fU* C*fU*fU*fC* A*fU*fC*fC* 138ps-rxRNA) U Key for Tables 3 and 4: f = 2′fluoro m = 2′Ome P = 5phosphate * = phosphorothioate linkage . = phosphodiester linkage DY547= DY547 dye d = deoxyribose

It should be appreciated that while some of the sequences presented inTables 1-8 are depicted as being attached to the dye DY547, all of thesequences presented in Tables 1-8 are also disclosed herein in theabsence of DY547.

Example 2: sd-rxRNA Variant Uptake in CNS

FIGS. 3-5 demonstrate that sd-rxRNA chemical variants, with varyinglevels of phosphorothioates, are taken up and delivered to cells in theCNS. To determine the tissue distribution of the sd-rxRNA chemicalvariants, fluorescently labeled compounds targeting SOD1 wereadministered to Sprague Dawley rats by intracisternal injection (ICinjection), with 15 μL of a 15 mg/mL solution. Twenty four hours postinjection tissues were harvested and processed for confocal microscopy.Confocal imaging was used to detect cellular uptake of sd-rxRNAvariants. The level of phosphorothioate content correlated with theobserved levels of cellular uptake in the CNS (e.g. increased levels ofphosphorothioate content resulted in greater uptake).

Example 3: sd-rxRNA Variant Silencing of SOD1 in CNS

FIG. 6 demonstrates SOD1 silencing in vivo (mouse, lumbar spinal cord(LSC)) following 14 day intrathecal administration of a SOD1 targetingsd-rxRNA variant (Oligo ID 25652). A 37% reduction of SOD1 mRNA levelswas observed in mice treated with the SOD1-targeting sd-rxRNA variantcompared to the non-targeting control (FIG. 6).

Methods: SOD1-targeting sd-rxRNA variant or non-targeting control (NTC)was administered by intrathecal infusion, using an osmotic pump (filledwith 100 μL of a 10 mg/mL solution of compound) for 14 days. Terminalbiopsy samples of the spinal cord were harvested on Day 14. RNA wasisolated and subjected to gene expression analysis by qPCR. Data werenormalized to the level of the cyclophilin B (PPIB) housekeeping geneand graphed relative to the non-targeting control set at 1.0. Error barsrepresent standard deviation between the individual biopsy samples. Pvalue for SOD1-targeting sd-rxRNA variant-treated group vs PBS groupwas * p<0.001.

Example 4: sd-rxRNA Variant Silencing of SOD1 in CNS

FIG. 7 demonstrates SOD1 silencing, in vivo (mouse, lumbar spinal cord(LSC)) following 14 day intrathecal administration of a SOD1 targetingsd-rxRNA variant (Oligo ID 25645). A statistically significant 24%reduction of SOD1 mRNA levels was observed in mice treated with theSOD1-targeting sd-rxRNA variant compared to the non-targeting control(FIG. 9).

Methods: SOD1-targeting sd-rxRNA variant or non-targeting control (NTC)was administered by intrathecal infusion, using an osmotic pump (filledwith 100 μL of a 10 mg/mL solution of compound) for 14 days. Terminalbiopsy samples of the spinal cord were harvested on Day 14. RNA wasisolated and subjected to gene expression analysis by qPCR. Data werenormalized to the level of the cyclophilin B (PPIB) housekeeping geneand graphed relative to the non-targeting control set at 1.0. Error barsrepresent standard deviation between the individual biopsy samples. Pvalue for SOD1-targeting sd-rxRNA variant-treated group vs non-targetingcontrol group was * p<0.01.

Example 5: Identification of SOD1-Targeting sd-rxRNA Variants

sd-rxRNA variants, containing hydrophobic modifications on position 4 or5 of the base, targeting SOD were designed, synthesized and screened invitro to determine the ability of the sd-rRNA variant to reduce targetgene mRNA levels. The sd-rxRNA variants were tested for activity in HeLacells (human cervical carcinoma cell line, 10,000 cells/well, 96 wellplate). HeLa cells were treated with varying concentrations of a panelof SOD1-targeting sd-rxRNAs variants or non-targeting control in serumcontaining media. Concentrations tested were 5, 1 and 0.1 μM. Thenon-targeting control sd-rxRNA is of similar structure to theSOD1-targeting sd-rxRNA variants and contains similar stabilizingmodifications throughout both strands. Forty eight hours postadministration, cells were lysed and mRNA levels determined by theQuantigene branched DNA assay according to the manufacture's protocolusing gene-specific probes (Affymetrix, Santa Clara, Calif.). FIGS. 8-11demonstrate the SOD1-targeting sd-rxRNA variants, containing hydrophobicmodifications on position 4 or 5 of the base, significantly reducetarget gene mRNA levels in vitro in HeLa cells. Sequences correspondingto FIGS. 8-11 can be found in Tables 5-8, respectively. Data werenormalized to a house keeping gene (PPIB) and graphed with respect tothe non-targeting control. Error bars represent the standard deviationfrom the mean of biological duplicates.

TABLE 5 SOD1 sd-rxRNA Variant Octyl Modifications SEQ SEQ ID Z=    Passenger Strand ID NO: Guide Strand ID NO: 25568 octylDY547.mA*mG* G*mZ* G* G* 139 P.mU*fU*fC* A*fU*fU*fU*fC*fC* 145 UA* A* A*mZ* G*mA*mA A*fC*mC*mU*mU*mU* G*mC*mC*fC* A* A 25569 octylDY547.mA*mG* G*mY* G* G* 140 P.mU*fU*fC* A*fU*fU*fU*fC*fC* 146 UA* A* A*mZ* G*mA*mA A*fC*mC*mU*mU*mU* G*mC*mC*fC* A* A 25570 octylDY547.mA*mG* G*mZ* G* G* 141 P.mU*fU*fC* A*fU*fU*fU*fC*fC* 147 UA* A* A*mY* G*mA*mA A*fC*mC*mU*mU*mU* G*mC*mC*fC* A* A 25571DY547.mA*mG* G*mY* G* G* 142 P.mU*fU*fC* A*fU*fU*fU*fC*fC* 148A* A* A*mY* G*mA*mA A*fC*mC*mU*mU*mU* G*mC*mC*fC* A* A 25572 octylDY547.mA*mG* G*mU* G* G* 143 P.mY*fZ*fX* A*fY*fY*fZ*fX*fX* 149 UA* A* A*mU* G*mA*mA A*fX*mX*mZ*mY*mY* G*mX*mX*fX* A* A 24560DY547.mA*mG* G*mU* G* G* 144 P.mU*fU*fC* A*fU*fU*fU*fC*fC* 150A* A* A*mU G*mA*mA A*fC*mC*mU*mU*mU* G*mC*mC*fC* A* A

TABLE 6 SOD1 sd-rxRNA Variant Octyl Modifications SEQ SEQ ID Z=    Passenger Strand ID NO: Guide Strand ID NO: 25573 octylDY547.mA*mG* G*mU* G* 151 P.mY*fY*fX* A*fY*fY*fZ*fX*fX* 157G* A* A* A*mU* G*mA*mA A*fX*mX*mZ*mY*mY* G*mX*mX*fX* A* A 25574 octylDY547.mA*mG* G*mU* G* 152 P.mY*fZ*fX* A*fY*fY*fY*fX*fX* 158G* A* A* A*mU* G*mA*mA A*fX*mX*mZ*mY*mY* G*mX*mX*fX* A* A 25575 octylDY547.mA*mG* G*mU* G* 153 P.mY*fZ*fX* A*fY*fY*fZ*fX*fX* 159G* A* A* A*mU* G*mA*mA A*fX*mX*mY*mY*mY* G*mX*mX*fX* A* A 25576DY547.mA*mG* G*mU* G* 154 P.mY*fY*fX* A*fY*fY*fY*fX*fX* 160G* A* A* A*mU* G*mA*mA A*fX*mX*mY*mY*mY* G*mX*mX*fX* A* A 25577thiophene DY547.mA*mG* G*mZ* G* 155 P.mU*fU*fC* A*fU*fU*fU*fC*fC* 161G* A* A* A*mZ* G*mA*mA A*fC*mC*mU*mU*mU* G*mC*mC*fC* A* A 24560DY547.mA*mG* G*mU* G* 156 P.mU*fU*fC* A*fU*fU*fU*fC*fC* 162G* A* A* A*mU* G*mA*mA A*fC*mC*mU*mU*mU* G*mC*mC*fC* A* A

TABLE 7 SOD1 sd-rxRNA Variant Thiophene Modifications SEQ SEQ ID Z=    Passenger Strand ID NO: Guide Strand ID NO: 25578 thiopheneDY547.mA*mG* G*mY* 163 P.mU*fU*fC A*fU*fU*fU*fC*fC* 169G* G* A* A* A*mZ* A*fC*mC*mU*mU*mU* G*mC*mC*fC* A* G*mA*mA A 25579thiophene DY547.mA*mG* G*mZ* 164 P.mU*fU*fC A*fU*fU*fU*fC*fC* 170G* G* A* A* A*mY* A*fC*mC*mU*mU*mU* G*mC*mC*fC* A* G*mA*mA A 25580DY547.mA*mG* G*mY* 165 P.mU*fU*fC A*fU*fU*fU*fC*fC* 171G* G* A* A* A*mY* A*fC*mC*mU*mU*mU* G*mC*mC*fC* A* G*mA*mA A 25584thiophene DY547.mA*mG* G*mU* 166 P.mY*fZ*fX* A*fY*fY*fZ*fX*fX* 172G* G* A* A* A*mU* A*fX*mX*mY*mY*mY* G*mX*mX*fX* A* A G*mA*mA 25585thiophene DY547.mA*mG* G*mU* 167 P.mY*fY*fX* A*fY*fY*fY*fX*X* 173G* G* A* A* A*mU* A*fX*mX*mY*mY*mY* G*mX*mX*fX* A* A G*mA*mA 24560DY547.mA*mG* G*mU* 168 P.mU*fU*fC* A*fU*fU*fU*fC*fC* 174G* G* A* A* A*mU* A*fC*mC*mU*mU*mU* G*mC*mC*fC* A* G*mA*mA A

TABLE 8 SOD1 sd-rxRNA Variant Isobutyl Modifications SEQ SEQ ID Z=    Passenger Strand ID NO: Guide Strand ID NO: 25586 isobutylDY547.mA*mG* G*dZ* G* 175 P.mU*fU*fC* A*fU*fU*fU*fC*fC* 181G* A* A* A*dZ* G*mA*mA A*fC*mC*mU*mU*mU* G*mC*mC*fC* A* A 25587 isobutylDY547.mA*mG* G*mY* G* 176 P.mU*fU*fC* A*fU*fU*fU*fC*fC* 182G* A* A* A*dZ* G*mA*mA a*fC*mC*mU*mU*mU* G*mC*mC*fC* A* A 25588 isobutylDY547.mA*mG* G*dZ* G* 177 P.mU*fU*fC* A*fU*fU*fU*fC*fC* 183G* A* A* A*mY* G*mA*mA A*fC*mC*mU*mU*mU* G*mC*mC*fC* A* A 25589 isobutylDY547.mA*mG* G*mU* G* 178 P.mY*dZ*fX* A*fY*fY*dZ*fX*fX* 184G* A* A* A*mU* G*mA*mA A*fX*mX*dZ*mY*mY* G*mX*mX*fX* A* A 25590 isobutylDY547.mA*mG* G*mU* G* 179 P.mY*fY*fX* A*fY*fY*dZ*fX*fX* 185G* A* A* A*mU* G*mA*mA A*fX*mX*dZ*mY*mY* G*mX*mX*fX* A* A 24560DY547.mA*mG* G*mU* G* 180 P.mU*fU*fC* A*fU*fU*fU*fC*fC* 186G* A* A* A*mU* G*mA*mA A*fC*mC*mU*mU*mU* G*mC*mC*fC* A* A Key for Tables5 through 8: X = 5 methyl C Y = 5 methyl U f = 2′fluoro m = 2′Ome P = 5phosphate * = phosphorothioate linkage . = phosphodiester linkage DY547= DY547 dye d = deoxyribose

EQUIVALENTS

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. Such equivalents areintended to be encompassed by the following claims.

All references, including patent documents, disclosed herein areincorporated by reference in their entirety. This applicationincorporates by reference the entire contents, including all thedrawings and all parts of the specification (including sequence listingor amino acid/polynucleotide sequences) of PCT Publication No.WO2010/033247 (Application No. PCT/US2009/005247), filed on Sep. 22,2009, and entitled “REDUCED SIZE SELF-DELIVERING RNAI COMPOUNDS,” U.S.Pat. No. 8,796,443, issued on Aug. 5, 2014, published as US 2012/0040459on Feb. 16, 2012, entitled “REDUCED SIZE SELF-DELIVERING RNAICOMPOUNDS,” U.S. Pat. No. 9,175,289, issued on Nov. 3, 2015, entitled“REDUCED SIZE SELF-DELIVERING RNAI COMPOUNDS,” PCT Publication No.WO2009/102427 (Application No. PCT/US2009/000852), filed on Feb. 11,2009, and entitled, “MODIFIED RNAI POLYNUCLEOTIDES AND USES THEREOF,”and US Patent Publication No. 2011/0039914, published on Feb. 17, 2011and entitled “MODIFIED RNAI POLYNUCLEOTIDES AND USES THEREOF,” PCTPublication No. WO 2011/119852, filed on Mar. 24, 2011 and entitled“REDUCED SIZE SELF-DELIVERING RNAI COMPOUNDS,” and U.S. Pat. No.9,080,171, issued on Jul. 14, 2015, and entitled “REDUCED SIZESELF-DELIVERING RNAI COMPOUNDS.”

1. An isolated double stranded nucleic acid molecule directed againstsuperoxide dismutase 1 (SOD1) comprising a guide strand and a passengerstrand, wherein the isolated double stranded nucleic acid moleculeincludes a double stranded region and a single stranded region, whereinthe region of the molecule that is double stranded is from 8-15nucleotides long, wherein the guide strand contains a single strandedregion that is 2-14 nucleotides long, wherein the guide strand contains2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21or 22 phosphorothioate modifications, wherein the passenger strand is 8to 15 nucleotides long, wherein the passenger strand contains 3, 4, 5,6, 7, 8, 9, 10, 11, 12, 13 or 14 phosphorothioate modifications, whereinat least 40% of the nucleotides of the isolated double stranded nucleicacid molecule are modified, and wherein the isolated double strandednucleic acid molecule comprises at least 12 contiguous nucleotides of asequence selected from the sequences within Tables 1-8, including themodification pattern provided in Tables 1-8.
 2. The isolated doublestranded nucleic acid molecule of claim 1, wherein at least 60% of thenucleotides are modified.
 3. The isolated double stranded nucleic acidmolecule of claim 1 or claim 2, wherein at least one of the nucleotidesof the isolated double stranded nucleic acid molecule that is modifiedcomprises a 2′O-methyl or a 2′-fluoro modification.
 4. The isolateddouble stranded nucleic acid molecule of any one of claims 1 to 3,wherein at least one strand of the isolated double stranded nucleic acidmolecule is completely phosphorothioated, or is completelyphosphorothioated with the exception of one residue.
 5. The isolateddouble stranded nucleic acid molecule of any one of claims 1 to 4,wherein a plurality of the U's and/or C's include a hydrophobicmodification, selected from the group consisting of methyl, isobutyl,octyl, imidazole or thiophene and wherein the modifications are locatedon positions 4 or 5 of U's and/or C's.
 6. An isolated double strandednucleic acid molecule that comprises at least 12 contiguous nucleotidesof a sequence selected from the sequences within Tables 1-8, wherein ifthe isolated double stranded nucleic acid molecule comprises at least 12contiguous nucleotides of a sequence selected from SEQ ID NOs: 70, 71,72, 73, 79, 80, 81, or 84 in Table 2, then the guide strand containsmore than 6 phosphorothioate modifications.
 7. The isolated doublestranded nucleic acid molecule of any one of claims 1 to 6, wherein theisolated double stranded nucleic acid molecule further comprises ahydrophobic conjugate that is attached to the isolated double strandednucleic acid molecule.
 8. The isolated double stranded nucleic acidmolecule of any one of claims 1 to 7, wherein the sense strand comprisesSEQ ID NO: 2, SEQ ID NO: 32, or SEQ ID NO: 122, and the guide strandcomprises SEQ ID NO: 61, SEQ ID NO: 91, or SEQ ID NO:
 123. 9. Theisolated double stranded nucleic acid molecule of any one of claims 1 to7, wherein the sense strand comprises SEQ ID NO: 4, SEQ ID NO: 34, orSEQ ID NO: 126, and the guide strand comprises SEQ ID NO: 63 or SEQ IDNO:
 93. 10. The isolated double stranded nucleic acid molecule of anyone of claims 1 to 7, wherein the sense strand comprises SEQ ID NO: 9,SEQ ID NO: 38, or SEQ ID NO: 135, and the guide strand comprises SEQ IDNO: 68, SEQ ID NO: 97, or SEQ ID NO:
 136. 11. The isolated doublestranded nucleic acid molecule of any one of claims 1 to 7, wherein thesense strand comprises SEQ ID NO: 10 or SEQ ID NO: 39, and the guidestrand comprises SEQ ID NO: 69 or SEQ ID NO:
 98. 12. The isolated doublestranded nucleic acid molecule of any one of claims 1 to 7, wherein thesense strand comprises SEQ ID NO: 5, SEQ ID NO: 127 or SEQ ID NO: 137,and the guide strand comprises SEQ ID NO: 64, SEQ ID NO: 128 or SEQ IDNO:
 138. 13. A composition comprising the isolated double strandednucleic acid molecule of any one of claims 1 to
 12. 14. The compositionof claim 13, further comprising a pharmaceutically acceptable carrier.15. The composition of claim 13 or claim 14 further comprising a secondtherapeutic agent.
 16. A method for treating ALS comprisingadministering to a subject in need thereof a therapeutically effectiveamount of an isolated double stranded nucleic acid molecule of any oneof claims 1 to 12, or a composition of any one of claims 13 to
 15. 17. Amethod for treating ALS comprising administering to a subject in needthereof a therapeutically effective amount of an isolated doublestranded nucleic acid molecule directed against superoxide dismutase 1(SOD1) comprising a guide strand and a passenger strand, wherein theisolated double stranded nucleic acid molecule includes a doublestranded region and a single stranded region, wherein the region of themolecule that is double stranded is from 8-15 nucleotides long, whereinthe guide strand contains a single stranded region that is 2-14nucleotides long, wherein the guide strand contains 2, 3, 4, 5, 6, 7, 8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 or 22 phosphorothioatemodifications, wherein the passenger strand is 8 to 15 nucleotides long,wherein the passenger strand contains 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13 or 14 phosphorothioate modifications, wherein at least 40% of thenucleotides of the isolated double stranded nucleic acid molecule aremodified, and wherein the isolated double stranded nucleic acid moleculecomprises at least 12 contiguous nucleotides of a sequence selected fromthe sequences within Tables 1-8, including the modification patternprovided in Tables 1-8.
 18. The method of claim 17, wherein the isolateddouble stranded nucleic acid molecule further comprises a hydrophobicconjugate that is attached to the isolated double stranded nucleic acidmolecule.
 19. The method of claim 17 or claim 18, wherein at least onestrand of the isolated double stranded nucleic acid molecule iscompletely phosphorothioated, or is completely phosphorothioated withthe exception of one residue.
 20. The method of any one of claims 17 to19, wherein the isolated double stranded nucleic acid molecule isformulated for delivery to the central nervous system.
 21. The method ofany one of claims 17 to 20, wherein the sense strand of the isolateddouble stranded nucleic acid molecule comprises SEQ ID NO: 2, SEQ ID NO:32, or SEQ ID NO: 122, and the guide strand of the isolated doublestranded nucleic acid molecule comprises SEQ ID NO: 61, SEQ ID NO: 91,or SEQ ID NO:
 123. 22. The method of any one of claims 17 to 20, whereinthe sense strand of the isolated double stranded nucleic acid moleculecomprises SEQ ID NO: 4, SEQ ID NO: 34, or SEQ ID NO: 126, and the guidestrand of the isolated double stranded nucleic acid molecule comprisesSEQ ID NO: 63 or SEQ ID NO:
 93. 23. The method of any one of claims 17to 20, wherein the sense strand of the isolated double stranded nucleicacid molecule comprises SEQ ID NO: 9, SEQ ID NO: 38, or SEQ ID NO:135,and the guide strand of the isolated double stranded nucleic acidmolecule comprises SEQ ID NO: 68, SEQ ID NO: 97, or SEQ ID NO:
 136. 24.The method of any one of claims 17 to 20, wherein the sense strand ofthe isolated double stranded nucleic acid molecule comprises SEQ ID NO:10 or SEQ ID NO: 39, and the guide strand of the isolated doublestranded nucleic acid molecule comprises SEQ ID NO: 69 or SEQ ID NO: 98.25. The method of any one of claims 17 to 20, wherein the sense strandof the isolated double stranded nucleic acid comprises SEQ ID NO: 5, SEQID NO: 127 or SEQ ID NO: 137, and the guide strand of the isolateddouble stranded nucleic acid comprises SEQ ID NO: 64, SEQ ID NO: 128 orSEQ ID NO:
 138. 26. The method of any one of claims 17 to 25, whereinthe isolated double stranded nucleic acid molecule is administered viaintrathecal infusion and/or injection.